Anti-IL12Rbeta1 antibodies and their use in treating autoimmune and inflammatory disorders

ABSTRACT

The present invention relates to antibodies that specifically bind to IL12Rβ1, the non-signal transducing chain of both the heterodimeric IL12 and IL23 receptors. The invention more specifically relates to specific antibodies that are IL12 and IL23 receptor antagonists capable of inhibiting IL12/IL18 induced IFNγ production of blood cells and compositions and methods of use for said antibodies to treat pathological disorders that can be treated by inhibiting IFNγ production, IL12 and/IL23 signaling, such as rheumatoid arthritis, psoriasis or inflammatory bowel diseases or other autoimmune and inflammatory disorders.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 13/252,721filed Oct. 4, 2011, which claims priority to U.S. ProvisionalApplication No. 61/389,916 filed Oct. 5, 2010, the contents of each ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2011 isnamed 54362.txt and is 139,264 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to antibodies that specifically bind toIL12Rβ1, the non-signal transducing chain of both the heterodimeric IL12and IL23 receptors.

The invention more specifically relates to specific antibodies that areIL12 and IL23 receptor antagonists capable of inhibiting IL12/IL18induced IFNγ production of blood cells and compositions and methods ofuse for said antibodies to treat pathological disorders that can betreated by inhibiting IFNγ production, IL12 and/IL23 signaling, such asrheumatoid arthritis, psoriasis or inflammatory bowel diseases or otherautoimmune and inflammatory disorders.

The IL12 receptor beta 1 (IL12Rβ1) chain is known as a potentialtherapeutic target for the treatment of Th1/Th17 mediated disorders,such as psoriasis and other autoimmune and inflammatory disorders.Psoriasis is a common chronic inflammatory skin disease characterized byhyper-proliferation of the epidermal layer and a prominent infiltrate ofdendritic cells and T cells. T cells play a key role in the pathologicalreactions occurring in the skin by secreting type 1 cytokines (includingIFN-γ and TNF-α) and that induce keratinocyte hyperproliferation,angiogenesis and neutrophil infiltration.

Two cytokines that are thought to be important in the development of Th1immune responses in psoriasis are interleukin-12 (IL12) andinterleukin-23 (IL23). Both cytokines are produced by antigen-presentingcells, such as macrophages and dendritic cells, and function byactivating T cells and natural killer cells. IL12 and IL23 are membersof a heterodimeric family of soluble cytokines that are comprised ofp35/p40 protein subunits in IL12 and p19/p40 protein subunits in IL23.The p40 subunit of either cytokine binds to the transmembrane IL12receptor β1 (IL12Rβ1) that is found on the surface of immune cells.Interruption of the IL12 p40/IL12Rβ1 interaction may prevent thebiological activity of both IL12 and IL23 (Presky et al., J. Immunol.160 (1998): 2174-79, Parham et al., J. Immunol. 168 (2002): 5699-5708.).

Several inflammatory and autoimmune diseases including psoriasis arelinked to exacerbated Th1 and/or Th17 responses. Many of them arecurrently treated either with general immuno-suppressants or veryselectively acting biologicals such as anti-TNF-α antibodies that arenot effective in all patients. These were found to increase the risk forinfections and to become ineffective after repeated treatment.Therefore, there is an unmet medical need for treatments with increasedsafety profiles and simultaneous capacity to induce long-term remissionor cure of the disease.

A neutralizing antibody to IL12p40 successfully abolished psoriaticlesions in mice, even when administered after transfer of the T cellsubset that induced the psoriasis-like condition (Hong et al., J.Immunol. 162.12 (1999): 7480-91.). An anti-IL12p40 antibody targetingboth IL12 and IL23 is currently in clinical trials for Psoriasis(Kauffman et al. J. Invest Dermatol. 123.6 (2004): 1037-44, Papp et al.Lancet. 371.9625 (2008): 1675-84, Kimball et al. Arch. Dermatol. 144.2(2008): 200-07), Crohn's Disease (Sandborn et al., Gastroenterology.135.4 (2008): 1130-41) and Multiple Sclerosis (Segal et al., LancetNeurol. 7.9 (2008): 796-804). Targeting IL12Rβ1 and hence,differentiation and maintenance of Th1 and Th17 cell populations as wellas the IL12 and IL23 mediated inflammatory cytokine production by thesecells, offers an opportunity for an improved therapeutic agent.

U.S. Pat. No. 6,046,012 refers to IL12Rβ1 and antibodies binding toanti-IL12Rβ1 in general. Anti-mouse IL12Rβ1 monoclonal antibodies arealso commercialized by Becton Dickinson (Cat#551455).

However, to date, there is no description in the art of bindingmolecules to human IL12Rβ1 showing highly potent IL12Rβ1 antagonisticactivity, for use in the treatment of autoimmune and inflammatorydisorders, such as psoriasis, rheumatoid arthritis or Crohn's disease.Only indirect evidence by targeting the respective interaction partner(IL12p40) validates the pathway.

Therefore, in one aspect, the invention provides an antibody, or aprotein comprising an antigen-binding portion of said antibody, thatbinds both to the IL12 receptor and the IL23 receptor, characterized inthat the antibody or protein specifically binds to IL12Rβ1 polypeptideof SEQ ID NO:89.

In one specific embodiment, the isolated antibody or protein of theinvention, comprises either

(a) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3 and a variable lightchain amino acid sequence comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQID NO:5 and LCDR3 of SEQ ID NO:6;

(b) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:7, HCDR2 of SEQ ID NO:8, HCDR3 of SEQ ID NO:9 and a variable lightchain amino acid sequence comprising LCDR1 of SEQ ID NO:10, LCDR2 of SEQID NO:11 and LCDR3 of SEQ ID NO:12;

(c) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:13, HCDR2 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15 and a variablelight chain amino acid sequence comprising LCDR1 of SEQ ID NO:16, LCDR2of SEQ ID NO:17 and LCDR3 of SEQ ID NO:18;

(d) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21 and a variablelight chain amino acid sequence comprising LCDR1 of SEQ ID NO:22, LCDR2of SEQ ID NO:23 and LCDR3 of SEQ ID NO:24;

(e) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:25, HCDR2′ of SEQ ID NO:26, HCDR3′ of SEQ ID NO:27 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:28,LCDR2′ of SEQ ID NO:29 and LCDR3′ of SEQ ID NO:30;

(f) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:31, HCDR2′ of SEQ ID NO:32, HCDR3′ of SEQ ID NO:33 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:34,LCDR2′ of SEQ ID NO:35 and LCDR3′ of SEQ ID NO:36;

(g) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:37, HCDR2′ of SEQ ID NO:38, HCDR3′ of SEQ ID NO:39 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:40,LCDR2′ of SEQ ID NO:41 and LCDR3′ of SEQ ID NO:42;

(h) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:43, HCDR2′ of SEQ ID NO:44, HCDR3′ of SEQ ID NO:45 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:46,LCDR2′ of SEQ ID NO:47 and LCDR3′ of SEQ ID NO:48; or,

(i) a variable heavy chain (V_(H)) and a variable light chain (V_(L))amino acid sequence where each of the CDRs share at least 60, 70, 80,90, 95 or 100 percent sequence identity with the corresponding CDRsequence of at least one antibody or protein described in (a)-(h) above,

and said antibody or protein binds to the IL12Rβ1 polypeptide of SEQ IDNO:89 with a K_(D) of 1 nM or less, 100 pM or less, or 10 pM or less andinhibits IL12 and/or IL23 binding to IL12Rβ1 polypeptide as measured inan in vitro competitive binding assay.

In another aspect the invention provides an isolated antibody or proteinwith an antigen-binding portion of an antibody which (i) binds to anepitope of the human IL12Rbeta1 polypeptide of SEQ ID NO:89, wherein theepitope: a) is comprised within amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide of SEQ ID NO:89; or b) comprises at leastone, two, three, four, five, six, seven, eight, or nine or more of theamino acid residues as defined in a) listed above; or c) comprises theamino acid residues as defined in a) listed above.

In one embodiment there is provided an isolated antibody or protein withan antigen-binding portion of an antibody which (i) binds to an epitopeof the human IL12Rbeta1 polypeptide of SEQ ID NO:89, wherein theepitope: a) is comprised within amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide of SEQ ID NO:89; or b) comprises at leastone, two, three, four, five, six, seven, eight, or nine or more of theamino acid residues as defined in a) listed above; or c) comprises theamino acid residues as defined in a) listed above, and/or (ii) competeswith an antibody which binds the epitope defined in (i) above.

In another embodiment there is provided an isolated antibody or proteinwith an antigen-binding portion of an antibody which (i) binds to anepitope of the human IL12Rbeta1 polypeptide of SEQ ID NO:89 as measuredusing amide Hydrogen/Deuterium exchange Mass Spectrometry, wherein theepitope: a) is comprised within amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide of SEQ ID NO:89; or b) comprises at leastone, two, three, four, five, six, seven, eight, or nine or more of theamino acid residues as defined in a) listed above; or c) comprises theamino acid residues as defined in a) listed above, and/or (ii) competeswith an antibody which binds the epitope defined in (i) above.

In another embodiment there is provided an isolated antibody or proteinwith an antigen-binding portion of an antibody which (i) binds to anepitope of the human IL12Rbeta1 polypeptide of SEQ ID NO:89, wherein theepitope: a) is comprised within amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide; or b) comprises at least one, two, three,four, five, six, seven, eight, or nine or more of the amino acidresidues as defined in a) listed above; or c) comprises the amino acidresidues as defined in a) listed above, and/or (ii) competes with anantibody which binds the epitope defined in (i) above, wherein saidantibody or protein binds to the IL12Rbeta1 polypeptide of SEQ ID NO:89with a KD of 1 nM or less and inhibits IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay.

In another embodiment there is provided an isolated antibody or proteinwith an antigen-binding portion of an antibody which (i) binds to anepitope of the human IL12Rbeta1 polypeptide of SEQ ID NO:89 as measuredusing amide Hydrogen/Deuterium exchange Mass Spectrometry, wherein theepitope: a) is comprised within amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide; or b) comprises at least one, two, three,four, five, six, seven, eight, or nine or more of the amino acidresidues as defined in a) listed above; or c) comprises the amino acidresidues as defined in a) listed above, and/or (ii) competes with anantibody which binds the epitope defined in (i) above, wherein saidantibody or protein binds to the IL12Rbeta1 polypeptide of SEQ ID NO:89with a KD of 1 nM or less and inhibits IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe human IL12Rbeta1 polypeptide of SEQ ID NO:89 and/or competes with anantibody or a protein with an antigen-binding portion of an antibodythat binds to an epitope within residues 416 to 429 of the humanIL12Rbeta1 polypeptide of SEQ ID NO:89.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe human IL12Rbeta1 polypeptide of SEQ ID NO:89 as measured using amideHydrogen/Deuterium exchange Mass Spectrometry, and/or competes with anantibody or a protein with an antigen-binding portion of an antibodythat binds to an epitope within residues 416 to 429 of the humanIL12Rbeta1 polypeptide of SEQ ID NO:89.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe human IL12Rbeta1 polypeptide of SEQ ID NO:89 and/or competes with anantibody or a protein with an antigen-binding portion of an antibodythat binds to an epitope within residues 416 to 429 of the humanIL12Rbeta1 polypeptide of SEQ ID NO:89, wherein said antibody or proteinbinds to the IL12Rbeta1 polypeptide of SEQ ID NO:89 with a KD of 1 nM orless and inhibits IL12 and/or IL23 binding to the IL12Rbeta1 polypeptideof SEQ ID NO:89 as measured in an in vitro competitive binding assay.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe human IL12Rbeta1 polypeptide of SEQ ID NO:89 as measured using amideHydrogen/Deuterium exchange Mass Spectrometry and/or competes with anantibody or a protein with an antigen-binding portion of an antibodythat binds to an epitope within residues 416 to 429 of the humanIL12Rbeta1 polypeptide of SEQ ID NO:89, wherein said antibody or proteinbinds to the IL12Rbeta1 polypeptide of SEQ ID NO:89 with a KD of 1 nM orless and inhibits IL12 and/or IL23 binding to the IL12Rbeta1 polypeptideof SEQ ID NO:89 as measured in an in vitro competitive binding assay.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe IL12Rbeta1 polypeptide of SEQ ID NO:89 and/or competes with anantibody which binds within residues 416 to 429 of the IL12Rbeta1polypeptide of SEQ ID NO:89.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds within residues 416 to 429 of the IL12Rbeta1polypeptide of SEQ ID NO:89 as measured using amide Hydrogen/Deuteriumexchange Mass Spectrometry and/or competes with an antibody which bindswithin residues 416 to 429 of the IL12Rbeta1 polypeptide of SEQ IDNO:89.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody, wherein saidantibody or protein binds to an epitope within residues 416 to 429 ofthe IL12Rbeta1 polypeptide of SEQ ID NO:89 and/or competes with anantibody which binds within residues 416 to 429 of the IL12Rbeta1polypeptide of SEQ ID NO:89, wherein said antibody or protein binds tothe IL12Rbeta1 polypeptide of SEQ ID NO:89 with a KD of 1 nM or less andinhibits IL12 and/or IL23 binding to the IL12Rbeta1 polypeptide of SEQID NO:89 as measured in an in vitro competitive binding assay.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody which binds to atleast one amino acid residue within residues 416 to 429 of theIL12Rbeta1 polypeptide of SEQ ID NO:89 and/or competes with an antibodywhich binds to at least one amino acid residue within residues 416 to429 of the IL12Rbeta1 polypeptide of SEQ ID NO:89.

In another embodiment there is provided an isolated antibody or aprotein with an antigen-binding portion of an antibody which binds to atleast one amino acid residue within residues 416 to 429 of theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured using amideHydrogen/Deuterium exchange Mass Spectrometry and/or competes with anantibody which binds to at least one amino acid residue within residues416 to 429 of the IL12Rbeta1 polypeptide of SEQ ID NO:89.

In the embodiments described herein, epitope binding may be determinedusing amide Hydrogen/Deuterium exchange Mass Spectrometry or otherconventional epitope mapping techniques known in the art.

In the embodiments described herein, antibody competition can bemeasured using a Biacore or Elisa-based cross-blocking assay, asdescribed herein, or using other cross-blocking assays known in the art.

In preferred embodiments the antibody or protein with an antigen-bindingportion of an antibody binds to human IL12Rbeta1 at an epitope asdefined in above.

Antibodies or proteins with an antigen-binding portion of an antibodyfalling within the scope of the invention can be identified by (i)screening for specificity for the human IL12Rbeta1 polypeptide of SEQ IDNO:89; (ii) optionally determining the affinity of the antibody; (iii)optionally assessing inhibition of IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay; and (iv) optionally assessing binding to orwithin the epitope defined by amino acid residues 416 to 429 of thehuman IL12Rbeta1 polypeptide of SEQ ID NO:89 using amideHydrogen/Deuterium exchange Mass Spectrometry or other conventionalepitope mapping techniques known in the art, and/or assessingcompetition with an antibody which binds the epitope as defined aboveusing a Biacore or Elisa-based cross-blocking assay, as describedherein, or using other cross-blocking assays known in the art. Afterscreening step (i) antibodies or proteins falling within the scope ofthe invention may be identified by performing one, or two, or three orfour of the following steps: (a) determining the affinity of theantibody; (b) assessing inhibition of IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay; (c) assessing binding to or within theepitope defined by amino acid residues 416 to 429 of the humanIL12Rbeta1 polypeptide of SEQ ID NO:89 using amide Hydrogen/Deuteriumexchange Mass Spectrometry or other conventional epitope mappingtechniques known in the art, and (d) assessing competition with anantibody which binds the epitope as defined above using a Biacore orElisa-based cross-blocking assay, as described herein, or using othercross-blocking assays known in the art.

Antibodies or proteins with an antigen-binding portion of an antibodyfalling within the scope of the invention can be identified by (i)screening for specificity for the human IL12Rbeta1 polypeptide of SEQ IDNO:89; (ii) determining the affinity of the antibody; (iii) assessinginhibition of IL12 and/or IL23 binding to the IL12Rbeta1 polypeptide ofSEQ ID NO:89 as measured in an in vitro competitive binding assay; and(iv) optionally assessing binding to or within the epitope defined byamino acid residues 416 to 429 of the human IL12Rbeta1 polypeptide ofSEQ ID NO:89 using amide Hydrogen/Deuterium exchange Mass Spectrometryor other conventional epitope mapping techniques known in the art,and/or assessing competition with an antibody which binds the epitope asdefined above using a Biacore or Elisa-based cross-blocking assay, asdescribed herein, or using other cross-blocking assays known in the art.

Antibodies or proteins with an antigen-binding portion of an antibodyfalling within the scope of the invention can be identified by (i)screening for specificity for the human IL12Rbeta1 polypeptide of SEQ IDNO:89; (ii) determining the affinity of the antibody; (iii) assessinginhibition of IL12 and/or IL23 binding to the IL12Rbeta1 polypeptide ofSEQ ID NO:89 as measured in an in vitro competitive binding assay; and(iv) assessing binding to or within the epitope defined by amino acidresidues 416 to 429 of the human IL12Rbeta1 polypeptide of SEQ ID NO:89using amide Hydrogen/Deuterium exchange Mass Spectrometry or otherconventional epitope mapping techniques known in the art, and/orassessing competition with an antibody which binds the epitope asdefined above using a Biacore or Elisa-based cross-blocking assay, asdescribed herein, or using other cross-blocking assays known in the art.

Antibodies or proteins with an antigen-binding portion of an antibodyfalling within the scope of the invention can be identified by (i)screening for specificity for the human IL12Rbeta1 polypeptide of SEQ IDNO:89; and (ii) assessing binding to or within the epitope defined byamino acid residues 416 to 429 of the human IL12Rbeta1 polypeptide ofSEQ ID NO:89 using amide Hydrogen/Deuterium exchange Mass Spectrometryor other conventional epitope mapping techniques known in the art,and/or assessing competition with an antibody which binds the epitope asdefined above using a Biacore or Elisa-based cross-blocking assay, asdescribed herein, or using other cross-blocking assays known in the art.

In one specific embodiment, the isolated antibody or protein inhibitsIL12 dependent IFN-γ production in human blood cells with an IC₅₀ around1 nM, 100 pM or 10 pM or less.

The isolated antibody or protein according to the invention may be afully human or humanized antibody. In one embodiment, it comprises amutant or chemically modified amino acid Fc region, wherein said mutantor chemical modification confers no or decreased ADCC activity to saidantibody when compared to a corresponding antibody with wild type Fcregion. In a specific embodiment, it is a mutant silent IgG1 antibody.

In another embodiment, the antibody or protein of the inventionessentially consists of a pegylated antigen-binding portion of anantibody which specifically binds to IL12Rβ1 polypeptide (SEQ ID NO:89).

In another embodiment, the antibody or protein of the inventioncomprises a variable heavy chain region of an antibody (V_(H)) with apolypeptide sequence having at least 95 or 100 percent sequence identityto at least one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53 and SEQ IDNO:55.

In another embodiment, the antibody or protein of the inventioncomprises a variable light chain region of an antibody (V_(L)) with apolypeptide sequence having at least 95 or 100 percent sequence identityto at least one of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ IDNO:56.

In a specific embodiment, an antibody according to the invention isselected from the group consisting of:

(a) mAb1 consisting of a heavy chain amino acid sequence of SEQ ID NO:57and a light chain amino acid sequence of SEQ ID NO:69;

(b) mAb2 consisting of heavy chain amino acid sequence of SEQ ID NO:61and light chain amino acid sequence of SEQ ID NO:69;

(c) mAb3 consisting of heavy chain amino acid sequence of SEQ ID NO:65and light chain amino acid sequence of SEQ ID NO:69;

(d) mAb4 consisting of heavy chain amino acid sequence of SEQ ID NO:58and light chain amino acid sequence of SEQ ID NO:70;

(e) mAb5 consisting of heavy chain amino acid sequence of SEQ ID NO:62and light chain amino acid sequence of SEQ ID NO:70;

(f) mAb6 consisting of heavy chain amino acid sequence of SEQ ID NO:66and light chain amino acid sequence of SEQ ID NO:70;

(g) mAb7 consisting of heavy chain amino acid sequence of SEQ ID NO:59and light chain amino acid sequence of SEQ ID NO:71;

(h) mAb8 consisting of heavy chain amino acid sequence of SEQ ID NO:63and light amino acid chain sequence of SEQ ID NO:71;

(i) mAb9 consisting of heavy chain amino acid sequence of SEQ ID NO:67and light chain amino acid sequence of SEQ ID NO:71;

(j) mAb10 consisting of heavy chain sequence of SEQ ID NO:60 and lightchain sequence of SEQ ID NO:72;

(k) mAb11 consisting of heavy chain sequence of SEQ ID NO:64 and lightchain sequence of SEQ ID NO:72; or,

(l) mAb12 consisting of heavy chain sequence of SEQ ID NO:68 and lightchain sequence of SEQ ID NO:72;

(m) mAb13 consisting of heavy chain sequence of SEQ ID NO:90 and lightchain sequence of SEQ ID NO:69;

(n) mAb14 consisting of heavy chain sequence of SEQ ID NO:91 and lightchain sequence of SEQ ID NO:70;

(o) mAb15 consisting of heavy chain sequence of SEQ ID NO:92 and lightchain sequence of SEQ ID NO:71; and,

(p) mAb16 consisting of heavy chain sequence of SEQ ID NO:93 and lightchain sequence of SEQ ID NO:72.

In another embodiment, the antibody or binding protein of the inventionis cross-blocked from binding to IL12Rβ1 by at least one of theantibodies mAb1-mAb16 defined above. Alternatively, the isolatedantibody or binding protein of the invention may be selected among thatwhich cross-blocks at least one of the antibodies mAb1-mAb16 frombinding to IL12Rβ1.

The invention further relates to the use of said antibody or protein ofthe invention, in particular mAb1 to mAb16 antibodies, for use as amedicament, more preferably, for the treatment of a pathologicaldisorder that is mediated by IL12Rβ1 or that can be treated byinhibiting IFNγ production, IL12 and/or IL23 signaling. In one specificembodiment, the antibodies or proteins of the invention may be used forthe treatment of autoimmune and inflammatory disorders, such asrheumatoid arthritis, psoriasis or inflammatory bowel diseases.

The invention also encompasses pharmaceutical compositions comprisingsaid antibody or proteins, in combination with one or more of apharmaceutically acceptable excipient, diluent or carrier. In onespecific embodiment, the pharmaceutical composition additionallycomprises one or more other active ingredients.

In one specific embodiment, said pharmaceutical composition is alyophilisate. In another specific embodiment, the pharmaceuticalcomposition is a liquid formulation comprising a therapeuticallyacceptable amount of an antibody or protein of the invention, preferablyprepared as a pre-filled syringe.

The invention also relates to the means for producing the antibodies orproteins of the invention. Such means include nucleic acid moleculesencoding at least the heavy and/or light variable region(s) of theantibody or protein of the invention or cloning expression vectorscomprising such nucleic acids, in particular, for the recombinantproduction of an antibody or protein according to the invention, forexample, mAb1-mAb16, in a host cell. In a specific embodiment, suchcloning or expression vector comprises at least one nucleic acidselected from the group consisting of SEQ ID NOs:73-88 and SEQ IDNOs:94-97. In another embodiment, it comprises either at least thefollowing coding sequences of heavy and light chain sequences of any oneof mAb1 to mAb16, operatively linked to suitable promoter sequences:

(a) mAb1: SEQ ID NO:73 and SEQ ID NO:85;

(b) mAb2: SEQ ID NO:77 and SEQ ID NO:85;

(c) mAb3: SEQ ID NO:81 and SEQ ID NO:85;

(d) mAb4: SEQ ID NO:74 and SEQ ID NO:86;

(e) mAb5: SEQ ID NO:78 and SEQ ID NO:86;

(f) mAb6: SEQ ID NO:82 and SEQ ID NO:86;

(g) mAb7: SEQ ID NO:75 and SEQ ID NO:87;

(h) mAb8: SEQ ID NO:79 and SEQ ID NO:87;

(i) mAb9: SEQ ID NO:83 and SEQ ID NO:87;

(j) mAb10: SEQ ID NO:76 and SEQ ID NO:88;

(k) mAb11: SEQ ID NO:80 and SEQ ID NO:88;

(l) mAb12: SEQ ID NO:84 and SEQ ID NO:88;

(m) mAb13: SEQ ID NO:94 and SEQ ID NO:85;

(n) mAb14: SEQ ID NO:95 and SEQ ID NO:86;

(o) mAb15: SEQ ID NO:96 and SEQ ID NO:87; or,

(p) mAb16: SEQ ID NO:97 and SEQ ID NO:88.

The invention further relates to a host cell comprising one or morecloning or expression vectors as described above and to the process forthe production of an antibody or protein of the invention, in particularmAb1-mAb16, said process comprising culturing the host cell, purifyingand recovering said antibody or protein.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” or “signaling activity” refers to abiochemical causal relationship generally initiated by a protein-proteininteraction such as binding of a growth factor to a receptor, resultingin transmission of a signal from one portion of a cell to anotherportion of a cell. In general, the transmission involves specificphosphorylation of one or more tyrosine, serine, or threonine residueson one or more proteins in the series of reactions causing signaltransduction. Penultimate processes typically include nuclear events,resulting in a change in gene expression.

The term IL12Rβ1 or IL12 receptor beta 1 refers to human IL12Rβ1 asdefined in SEQ ID NO: 89, unless otherwise described.

The term p40 refers to human p40 subunit of human IL12 cytokine asdefined in SEQ ID NO: 99, unless otherwise described.

The term p35 refers to human p35 subunit of human IL12 cytokine asdefined in SEQ ID NO:100, unless otherwise described.

The term p19 refers to human p19 subunit of human IL23 cytokine asdefined in SEQ ID NO:101, unless otherwise described.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragments (i.e., “antigen-binding portion”) orsingle chains thereof.

A naturally occurring “antibody” is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a portion of IL12Rβ1). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR), or any fusion proteins comprising such antigen-bindingportion.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single chain protein in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toIL12Rβ1 is substantially free of antibodies that specifically bind toother antigens than IL12Rβ1). An isolated antibody that specificallybinds to IL12Rβ1 may, however, have cross-reactivity to other antigens,such as IL12Rβ1 molecules from other species. Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutantversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis, forexample, as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG4) that is provided by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody or a protein that “specifically binds toIL12Rβ1 polypeptide” is intended to refer to an antibody or protein thatbinds to human IL12Rβ1 polypeptide with a K_(D) of 100 nM or less, 10 nMor less, 1 nM or less, 100 pM or less, or 10 pM or less. An antibodythat “cross-reacts with an antigen other than IL12Rβ1” is intended torefer to an antibody that binds that antigen with a K_(D) of 10 nM orless, 1 nM or less, or 100 pM or less. An antibody that “does notcross-react with a particular antigen” is intended to refer to anantibody that binds to that antigen, with a K_(D) of 100 nM or greater,or a K_(D) of 1 μM or grater, or a K_(D) of 10 μM or greater. In certainembodiments, such antibodies that do not cross-react with the antigenexhibit essentially undetectable binding against these proteins instandard binding assays.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system. An assay for measuring anti-IL12Rβ1antibody K_(D) with the Biacore® system is described in the Examplesbelow.

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with the antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

As used herein, an antibody or protein that inhibits IL12 and/or IL23binding to IL12Rβ1 polypeptide is intended to refer to an antibody orprotein that inhibits IL12 and/or IL23 binding to IL12Rβ1 polypeptidewith an IC₅₀ of 10 nM or less, preferably with an IC₅₀ of 1 nM or less,more preferably with an IC₅₀ of 100 pM, or less, as measured in an invitro competitive binding assay such as Bioveris™ assay. Such assay isdescribed in more details in the examples below.

As used herein, the term “IL12R antagonist” is intended to refer to anantibody or protein that inhibits IL12Rβ1 induced signaling activity inthe presence of IL12 in a human cell assay such as the IL12 dependentIFNγ production assay in human blood cells. Such assay is described inmore details in the examples below. In some embodiments, the antibodiesor proteins of the invention inhibit IL12 dependent IFNγ production asmeasured in a human blood cell assay at an IC₅₀ of 10 nM or less, 1 nMor less, or 100 pM or less. Such assay is described in more details inthe examples below.

As used herein, the term “IL23R antagonist” is intended to refer to anantibody that inhibits IL12Rβ1 induced signaling activity in thepresence of IL23 in a human cell assay such as the IL23 dependent IFNγproduction assay in human blood cell. Such assay is described in moredetails in the examples below. In some embodiments, the antibodies orbinding protein of the invention inhibit IL23 IFNγ production asmeasured in a human blood cell assay at an IC₅₀ of 10 nM or less, 1 nMor less, or 100 pM or less. Such assay is described in more details inthe examples below.

As used herein, an antibody with “no agonistic activity” is intended torefer to an antibody that does not significantly increase IL12Rβ1mediated signaling activity in the absence of IL12 in a cell-basedassay, such as the IL12 IFNγ production assay in human blood cell. Suchassay is described in more details in the examples below.

As used herein, an antibody or protein that inhibits IL12 ex vivo IFNγproduction in whole blood cell is intended to refer to an antibody orprotein that decreases IL12 ex vivo IFNγ production to a level below 10%of the control level with an anti-IL12Rβ1 mAb plasma level above 10μg/ml. In some embodiments, it refers to antibodies or proteins thatcompletely abolish IL12 ex vivo IFNγ production in primate blood cellwith anti-IL12Rβ1 mAb plasma levels above 10 μg/ml. Such assays aredescribed in more details in the examples below.

As used herein, the term “ADCC” or “antibody dependent cellcytotoxicity” activity refers to cell depleting activity. ADCC activitycan be measured by the ADCC assay as described in more details in theExamples below.

As used herein, the term “selectivity” for an antibody or protein of theinvention refers to an antibody or protein that binds to a certaintarget polypeptide but not to closely related polypeptides.

As used herein, the term “high affinity” for an antibody refers to anantibody having a K_(D) of 1 nM or less for a target antigen. As usedherein, the term “subject” includes any human or nonhuman animal.

The term “nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows,chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence. The optimizedsequences herein have been engineered to have codons that are preferredin CHO mammalian cells, however optimized expression of these sequencesin other eukaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i. e., % identity=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. Alternatively, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The percent identity between two nucleotide amino acid sequences mayalso be determined using for example algorithms such as the BLASTNprogram for nucleic acid sequences using as defaults a word length (W)of 11, an expectation (E) of 10, M=5, N=4, and a comparison of bothstrands.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to IL12Rβ1 in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to IL12Rβ1, and therefore whether it can be said to cross-blockaccording to the invention, can be determined using standard competitionbinding assays. One suitable assay involves the use of the Biacoretechnology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala,Sweden)), which can measure the extent of interactions using surfaceplasmon resonance technology. Another assay for measuring cross-blockinguses an ELISA-based approach. Further details on these methods are givenin the Examples.

According to the invention, a cross-blocking antibody or other bindingagent according to the invention binds to IL12Rβ1 in the describedBIAcore cross-blocking assay such that the recorded binding of thecombination (mixture) of the antibodies or binding agents is between 80%and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding,specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximumtheoretical binding, and more specifically between 70% and 0.1% (e.g.70% to 4%), and more specifically between 65% and 0.1% (e.g. 65% to 4%)of maximum theoretical binding (as defined above) of the two antibodiesor binding agents in combination

An antibody is defined as cross-blocking in the ELISA assay as describedin the Examples, if the solution phase anti-IL12Rβ1 antibody is able tocause a reduction of between 60% and 100%, specifically between 70% and100%, and more specifically between 80% and 100%, of the IL12Rβ1detection signal (i.e. the amount of IL12Rβ1 bound by the coatedantibody) as compared to the IL12Rβ1 detection signal obtained in theabsence of the solution phase anti-IL12Rβ1 antibody (i.e. the positivecontrol wells).

Recombinant Antibodies

Antibodies of the invention include the human recombinant antibodiesmAb1-mAb16, isolated and structurally characterized by their full lengthheavy and light chain amino acid sequences as described in the Table 1below:

TABLE 1 Full length heavy and light chain amino acid sequences ofmAb1-mAb16 Full Length Heavy Chain Full Length Light Chain AntibodyAmino acid sequence Amino acid sequence mAb1 SEQ ID NO: 57 SEQ ID NO: 69mAb2 SEQ ID NO: 61 SEQ ID NO: 69 mAb3 SEQ ID NO: 65 SEQ ID NO: 69 mAb4SEQ ID NO: 58 SEQ ID NO: 70 mAb5 SEQ ID NO: 62 SEQ ID NO: 70 mAb6 SEQ IDNO: 66 SEQ ID NO: 70 mAb7 SEQ ID NO: 59 SEQ ID NO: 71 mAb8 SEQ ID NO: 63SEQ ID NO: 71 mAb9 SEQ ID NO: 67 SEQ ID NO: 71 mAb10 SEQ ID NO: 60 SEQID NO: 72 mAb11 SEQ ID NO: 64 SEQ ID NO: 72 mAb12 SEQ ID NO: 68 SEQ IDNO: 72 mAb13 SEQ ID NO: 90 SEQ ID NO: 69 mAb14 SEQ ID NO: 91 SEQ ID NO:70 mAb15 SEQ ID NO: 92 SEQ ID NO: 71 mAb16 SEQ ID NO: 93 SEQ ID NO: 72

The corresponding variable regions, V_(H) and V_(L) amino acid sequencesof such isolated antibodies mAb1-mAb16 of the invention are shown in theTable 2 below.

TABLE 2 Variable heavy and light chain amino acid sequences ofmAb1-mAb16 Variable Heavy Chain Variable Light Chain Antibody Amino acidsequence Amino acid sequence mAb1, mAb2, mAb3, SEQ ID NO: 49 SEQ ID NO:50 mAb13 mAb4, mAb5, mAb6, SEQ ID NO: 51 SEQ ID NO: 52 mAb14 mAb7, mAb8,mAb9, SEQ ID NO: 53 SEQ ID NO: 54 mAb15 mAb10, mAb11, SEQ ID NO: 55 SEQID NO: 56 mAb12, mAb16

Other antibodies of the invention include those having amino acids thathave been mutated by amino acid deletion, insertion or substitution, yethave at least 60, 70, 80, 90, 95 or 100 percent identity in the CDRregions with the CDR regions depicted in the sequences described above.In some embodiments, the antibody of the invention is a mutant variantof any one of mAb1-mAb16, wherein said mutant variant antibody includemutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 aminoacids have been mutated by amino acid deletion, insertion orsubstitution in the CDR regions when compared with the CDR regionsdepicted in the sequences described above.

Full length light and heavy chains nucleotide coding sequences ofmAb1-mAb16 are shown in the Table 3 below.

TABLE 3 Full length heavy and light chain DNA coding sequences FullLength Heavy Chain Full Length Light Chain Antibody DNA coding sequenceDNA coding sequence mAb1 SEQ ID NO: 73 SEQ ID NO: 85 mAb2 SEQ ID NO: 77SEQ ID NO: 85 mAb3 SEQ ID NO: 81 SEQ ID NO: 85 mAb4 SEQ ID NO: 74 SEQ IDNO: 86 mAb5 SEQ ID NO: 78 SEQ ID NO: 86 mAb6 SEQ ID NO: 82 SEQ ID NO: 86mAb7 SEQ ID NO: 75 SEQ ID NO: 87 mAb8 SEQ ID NO: 79 SEQ ID NO: 87 mAb9SEQ ID NO: 83 SEQ ID NO: 87 mAb10 SEQ ID NO: 76 SEQ ID NO: 88 mAb11 SEQID NO: 80 SEQ ID NO: 88 mAb12 SEQ ID NO: 84 SEQ ID NO: 88 mAb13 SEQ IDNO: 94 SEQ ID NO: 85 mAb14 SEQ ID NO: 95 SEQ ID NO: 86 mAb15 SEQ ID NO:96 SEQ ID NO: 87 mAb16 SEQ ID NO: 97 SEQ ID NO: 88

Other nucleic acids encoding antibodies of the invention include nucleicacids that have been mutated by nucleotide deletion, insertion orsubstitution, yet have at least 60, 70, 80, 90, 95 or 100 percentidentity to the CDR corresponding coding regions depicted in thesequences described above or in Tables 4 and 5 below.

In some embodiments, it include variant nucleic acids wherein no morethan 1, 2, 3, 4 or 5 nucleotides have been changed by nucleotidedeletion, insertion or substitution in the CDR coding regions with theCDR coding regions depicted in the sequences described above or inTables 4 and 5 below.

For antibodies that bind to the same epitope, the V_(H), V_(L), fulllength light chain, and full length heavy chain sequences (nucleotidesequences and amino acid sequences) can be “mixed and matched” to createother anti-IL12Rβ1 binding molecules of the invention. IL12Rβ1 bindingof such “mixed and matched” antibodies can be tested using the bindingassays described above or other conventional binding assays (e.g.,ELISAs). When these chains are mixed and matched, a V_(H) sequence froma particular V_(H)/V_(L) pairing should be replaced with a structurallysimilar V_(H) sequence. Likewise a full length heavy chain sequence froma particular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a V_(L) sequence from a particular V_(H)/V_(L)pairing should be replaced with a structurally similar V_(L) sequence.Likewise a full length light chain sequence from a particular fulllength heavy chain/full length light chain pairing should be replacedwith a structurally similar full length light chain sequence.Accordingly, in one aspect, the invention provides an isolatedrecombinant antibody having: a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:49, 51, 53 and 55; and a light chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 50, 52,54 and 56; wherein said heavy and light chain regions are selected suchthat the antibody specifically binds to IL12Rβ1.

Examples of the amino acid sequences of the V_(H) CDR1s (also calledHCDR1 or HCDR1′ depending of the CDR definition that is used), V_(H)CDR2s (also called HCDR2 or HCDR2′ depending of the CDR definition thatis used), V_(H) CDR3s (also called HCDR1 or HCDR1′ depending of the CDRdefinition that is used), V_(L) CDR1s (also called LCDR1 or LCDR1′depending of the CDR definition that is used), V_(L) CDR2s (also calledLCDR2 or LCDR2′ depending of the CDR definition that is used), V_(L)CDR3s (also called HCDR3 or HCDR3′ depending of the CDR definition thatis used) of some antibodies according to the invention are shown inTables 4 and 5 below.

In Table 4, the CDR regions of some antibodies of the invention aredelineated using the Kabat system (Kabat, E. A., et al., 1991 Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, see alsoZhao&Lu, 2009, Molecular Immunology 47: 694-700)

For the ease of reading, when CDR regions are delineated according toKabat definition, they are called hereafter HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, LCDR3 respectively.

TABLE 4 CDR regions of mAb1 to mAb16 according to Kabat definitionOriginal antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 mAb1, SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID mAb2, NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6mAb3, mAb13 mAb4, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb5, NO: 7NO: 8 NO: 9 NO: 10 NO: 11 NO: 12 mAb6, mAb14 mAb7, SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID mAb8, NO: 13 NO: 14 NO: 15 NO: 16 NO: 17 NO: 18mAb9, mAb15 mAb10, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb11, NO:19 NO: 20 NO: 21 NO: 22 NO: 23 NO: 24 mAb12, mAb16

In Table 5, the CDR regions of some antibodies of the invention aredelineated using the Chothia system, Al-Lazikani et al. 1997. J. Mol.Biol. 273, 927-948). For ease of reading, when the CDR regions aredelineated according to Chothia definition, they are called hereafterHCDR1′, HCDR2′, HCDR3′, LCDR1′, LCDR2′, LCDR3′ respectively.

TABLE 5 CDR regions from mAb1 to mAb16 according to Chothia definitionOriginal anti- body HCDR1′ HCDR2′ HCDR3′ LCDR1′ LCDR2′ LCDR3′ mAb1, SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb2, NO: 25 NO: 26 NO: 27 NO: 28NO: 29 NO: 30 mAb3, mAb13 mAb4, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID mAb5, NO: 31 NO: 32 NO: 33 NO: 34 NO: 35 NO: 36 mAb6, mAb14 mAb7, SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb8, NO: 37 NO: 38 NO: 39 NO: 40NO: 41 NO: 42 mAb9, mAb15 mAb10, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID mAb11, NO: 43 NO: 44 NO: 45 NO: 46 NO: 47 NO: 48 mAb12, mAb16

Given that each of these antibodies can bind to IL12Rβ1 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and matched, each antibody containing a V_(H)CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 create other anti-IL12Rβ1binding molecules of the invention). IL12Rβ1 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples or other conventional assays (e.g., ELISAs).When V_(H) CDR sequences are mixed and matched, the CDR1, CDR2 and/orCDR3 sequence from a particular V_(H) sequence should be replaced with astructurally similar CDR sequence(s). Likewise, when V_(L) CDR sequencesare mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from aparticular V_(L) sequence should be replaced with a structurally similarCDR sequence(s). It will be readily apparent to the ordinarily skilledartisan that novel V_(H) and V_(L) sequences can be created bysubstituting one or more V_(H) and/or V_(L) CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present invention.

In one embodiment, an isolated recombinant antibody, or a proteincomprising an antigen binding region thereof, has: a heavy chainvariable region CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 7, 13 and 19; a heavy chain variableregion CDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 8, 14 and 20; a heavy chain variable regionCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 9, 15 and 21; a light chain variable regionCDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 10, 16 and 22; a light chain variableregion CDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 11, 17 and 23; and a light chain variableregion CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 12, 18 and 24; wherein said CDR regions areselected so that the antibody or protein of the invention specificallybinds to IL12Rβ1.

In another embodiment, an isolated recombinant antibody, or a proteincomprising an antigen binding region thereof has: a heavy chain variableregion HCDR1′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 25, 31, 37 and 43; a heavy chain variableregion HCDR2′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 26, 32, 38 and 44; a heavy chain variableregion HCDR3′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 27, 33, 39 and 45; a light chain variableregion LCDR1′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 28, 34, 40 and 46; a light chain variableregion LCDR2′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 29, 35, 41 and 47; and a light chain variableregion LCDR3′ comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 30, 36, 42 and 48; wherein said CDR regionsare selected so that the antibody or protein of the inventionspecifically binds to IL12Rβ1.

In certain embodiments, the antibody or protein of the inventioncomprises either

(a) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3 and a variable lightchain amino acid sequence comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQID NO:5 and LCDR3 of SEQ ID NO:6;

(b) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:7, HCDR2 of SEQ ID NO:8, HCDR3 of SEQ ID NO:9 and a variable lightchain amino acid sequence comprising LCDR1 of SEQ ID NO:10, LCDR2 of SEQID NO:11 and LCDR3 of SEQ ID NO:12;

(c) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:13, HCDR2 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15 and a variablelight chain amino acid sequence comprising LCDR1 of SEQ ID NO:16, LCDR2of SEQ ID NO:17 and LCDR3 of SEQ ID NO:18;

(d) a variable heavy chain amino acid sequence comprising HCDR1 of SEQID NO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21 and a variablelight chain amino acid sequence comprising LCDR1 of SEQ ID NO:22, LCDR2of SEQ ID NO:23 and LCDR3 of SEQ ID NO:24;

(e) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:25, HCDR2′ of SEQ ID NO:26, HCDR3′ of SEQ ID NO:27 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:28,LCDR2′ of SEQ ID NO:29 and LCDR3′ of SEQ ID NO:30;

(f) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:31, HCDR2′ of SEQ ID NO:32, HCDR3′ of SEQ ID NO:33 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:34,LCDR2′ of SEQ ID NO:35 and LCDR3′ of SEQ ID NO:36;

(g) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:37, HCDR2′ of SEQ ID NO:38, HCDR3′ of SEQ ID NO:39 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:40,LCDR2′ of SEQ ID NO:41 and LCDR3′ of SEQ ID NO:42; or,

(h) a variable heavy chain amino acid sequence comprising HCDR1′ of SEQID NO:43, HCDR2′ of SEQ ID NO:44, HCDR3′ of SEQ ID NO:45 and a variablelight chain amino acid sequence comprising LCDR1′ of SEQ ID NO:46,LCDR2′ of SEQ ID NO:47 and LCDR3′ of SEQ ID NO:48.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody or protein of the invention hasfull length heavy and light chain amino acid sequences; full lengthheavy and light chain nucleotide sequences, variable region heavy andlight chain nucleotide sequences, or variable region heavy and lightchain amino acid sequences, or all 6 CDR regions amino acid sequences ornucleotide coding sequences that are homologous to the amino acid ornucleotide sequences of the antibodies mAb1-mAb16 described above, inparticular in Table 1, and wherein the antibodies or proteins of theinvention retain the desired functional properties of the originalmAb1-mAb16 antibodies.

Desired functional properties of the original mAb1-mAb16 antibodies maybe selected from the group consisting of:

-   -   (i) the binding affinity to IL12Rβ1 (specific binding to        IL12Rβ1), for example, a K_(D) being 1 nM or less, 100 pM or        less, or 10 pM or less, as measured in the Biacore assay        described in the Examples;    -   (ii) competitive inhibition of IL12 or IL23 binding to IL12Rβ1,        for example, an IC₅₀ being 10 nM or less, or 1 nM or less, or        100 pM or less, as measured in an IL12 or IL23 in vitro        competitive binding assay as described in the Examples;    -   (iii) IL12 and/or IL23 dependent inhibition of IFNγ production        in human blood cell, for example, an IC₅₀ being 10 nM or less,        or 1 nM or less, or 100 pM or less, as measured in an IL12 or        IL23 dependent IFNγ production in human blood cell assay as        described in the Examples;    -   (iv) IL12 dependent inhibition of ex vivo IFN-γ production in        primate blood cells;    -   (v) cross-reactivity with cynomolgous IL12Rβ1 polypeptide of SEQ        ID NO:98;    -   (vi) suitable properties for drug development, in particular, it        does not aggregate at in a formulation at high concentration,        ie, above 50 mg/ml; and,    -   (vii) it has no or low ADCC activity.

For example, the invention relates to homologous antibodies ofmAb1-mAb16 (or a binding protein comprising an antigen binding portionthereof), comprising a variable heavy chain (V_(H)) and a variable lightchain (V_(L)) sequences where the CDR sequences, i.e. the 6 CDR regions;HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 or HCDR1′, HCDR2′, HCDR3′,LCDR1′, LCDR2′, LCDR3′, share at least 60, 70, 90, 95 or 100 percentsequence identity to the corresponding CDR sequences of at least oneantibody of mAb1-mAb16, wherein said homologous antibody or bindingprotein specifically binds to IL12Rβ1, and the antibody or bindingprotein exhibits at least one of the following functional properties: itinhibits IL12 and IL23 binding to IL12Rβ1, it inhibits IL12 dependentIFNγ production in human blood cells, it inhibits IL23 dependent IFNγproduction in human blood cells, or it inhibits IL12 ex vivo IFN-γproduction in primate blood cells. In a related specific embodiment, thehomologous antibody or binding protein binds to IL12Rβ1 with a K_(D) of1 nM or less and inhibits IL12 and/or IL23 binding to IL12Rβ1 asmeasured in an in vitro competitive binding assay such as Bioveris™assay with an IC₅₀ of 1 nM or less. The CDRs of mAb1-mAb16 are definedin the above Tables 4 and 5.

The invention further relates to homologous antibodies of mAb1-mAb16 (ora binding protein comprising an antigen binding portion thereof)comprising a heavy chain variable region and a light chain variableregion that are at least 80%, 90%, or at least 95% or 100% identical tothe corresponding heavy and light chain variable regions of any one ofmAb1-mAb16 antibodies; the homologous antibody or binding proteinspecifically binds to IL12Rβ1, and it exhibits at least one of thefollowing functional properties: it inhibits IL12 and IL23 binding toIL12Rβ1, it inhibits IL12 dependent IFNγ production in human blood cell,it inhibits IL23 dependent IFNγ production in human blood cells, or itinhibits IL12 ex vivo IFN-γ production in primate blood cells. In arelated specific embodiment, the homologous antibody or binding proteinbinds to IL12Rβ1 with a K_(D) of 1 nM or less and inhibits IL12 and/orIL23 binding to IL12Rβ1 as measured in an in vitro competitive bindingassay such as Bioveris™ assay with an IC₅₀ of 1 nM or less. The V_(H)and V_(L) amino acid sequences of mAb1-mAb16 are defined in the abovetable 2.

In another example, the invention relates to homologous antibodies ofmAb1-mAb16 (or a binding protein comprising an antigen binding portionthereof) comprising a full length heavy chain and a full length lightchain, wherein: the variable heavy chain is encoded by a nucleotidesequence that is at least 80%, at least 90%, at least 95%, or 100%identical to the corresponding coding nucleotide sequence of thevariable heavy and light chains of mAb1-mAb16, the homologous antibodyor binding protein specifically binds to IL12Rβ1, and it exhibits atleast one of the following functional properties: it inhibits IL12 andIL23 binding to IL12Rβ1, it inhibits IL12 dependent IFNγ production inhuman blood cells, it inhibits IL23 dependent IFNγ production in humanblood cells, or it inhibits IL12 ex vivo IFN-γ production in primateblood cells. In a related specific embodiment, the homologous antibodyor binding protein binds to IL12Rβ1 with a K_(D) of 1 nM or less andinhibits IL12 and/or IL23 binding to IL12Rβ1 as measured in an in vitrocompetitive binding assay such as Bioveris™ assay with an IC₅₀ of 1 nMor less. The coding nucleotide sequences of the variable regions of mAb1to mAb16 can be derived from the Table 3 showing the full length codingnucleotide sequences of mAb1-mAb16 and Table 2 showing the amino acidsequences of the variable regions of mAb1-mAb16.

In various embodiments, the antibody or binding protein comprising anantigen-binding portion of an antibody, may exhibit one or more, two ormore, three or more, or four or more of the desired functionalproperties discussed above. The antibody or protein of the invention canbe, for example, a human antibody, a humanized antibody or a chimericantibody. Preferably the antibody or protein is a fully human silentantibody, preferably a fully human silent IgG1 antibody.

As used herein, the term “silent” antibody refers to an antibody thatexhibits no or low ADCC activity as measured in an ADCC activity assayas described in the Examples.

In one embodiment, the term “no or low ADCC activity” means that thesilent antibody exhibit an ADCC activity that is at below 50%, forexample below 10% of the ADCC activity that is observed with thecorresponding wild type (non silent) antibody.

Silenced effector functions can be obtained by mutation in the Fcconstant part of the antibodies and have been described in the Art:Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al., J.Immunol. 181 (2008): 6664-69, Strohl, Colo. Biotechnology 20 (2009):685-91). Examples of silent IgG1 antibodies comprise the so-called LALAmutant comprising L234A and L235A mutation in the IgG1 Fc amino acidsequence. Another example of a silent IgG1 antibody comprises the D265Amutation. Another silent IgG1 antibody comprises the N297A mutation,which results in aglycosylated or non-glycosylated antibodies.

Antibodies with mutant amino acid sequences can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of thecoding nucleic acid molecules, followed by testing of the encodedaltered antibody for retained function (i. e., the functions set forthabove) using the functional assays described herein.

Antibodies with Conservative Modifications

In certain embodiments, an antibody (or a binding protein comprisingantigen binding portion thereof) of the invention has a heavy chainvariable region comprising HCDR1, HCDR2, and HCDR3 sequences (or HCDR1′,HCDR2′ and HCDR3′) and a light chain variable region comprising LCDR1,LCDR2, and LCDR3 sequences (or LCDR1′, LCDR2′, and LCDR3′), wherein oneor more of these CDR sequences have specified amino acid sequences basedon the mAb1 to mAb16 antibodies described herein or conservativemodifications thereof, and wherein the antibody or protein retains thedesired functional properties of the anti-IL12Rβ1 antibodies of theinvention.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid substitutions in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Modifications can be introduced into an antibody of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis.

Antibodies that Cross-Block any One of mAb1-mAb16 and/or that Bind tothe Same Epitope as mAb1-mAb16

The antibodies mAb1-mAb16 have been shown to cross-compete at eachother. Therefore, additional antibodies can therefore be identifiedbased on their ability to cross-compete (e.g., to competitively inhibitthe binding of), in a statistically significant manner with otherantibodies of the invention, for example mAb1-mAb16, in standard IL12Rβ1binding assays. Test antibody may first be screened for their bindingaffinity to IL12Rβ1, for example from human recombinant antibodylibraries, using for example phage display technologies as describedbelow. The ability of a test antibody to cross-compete with or inhibitthe binding of antibodies of the present invention to human IL12Rβ1demonstrates that the test antibody can compete with that antibody forbinding to human IL12Rβ1; such an antibody may, according tonon-limiting theory, bind to the same or a related (e.g., a structurallysimilar or spatially proximal) epitope on human IL12Rβ1 as the antibodywith which it competes. Examples of Biacore or Elisa-basedcross-blocking assays are described in detail in the Examples.

Accordingly, in one embodiment, the invention provides an isolatedantibody or protein which cross-blocks or is cross-blocked by at leastone antibody of mAb1-mAb16, from binding to IL12Rβ1, wherein saidantibody or protein:

-   -   (i) binds to the IL12Rβ1 polypeptide of SEQ ID NO:89 with a        K_(D) of 1 nM or less, and (ii) inhibits IL12 and/or IL23        binding to the IL12Rβ1 polypeptide of SEQ ID NO:89 as measured        in an in vitro competitive binding assay.

In one specific embodiment, such cross-blocking anti-IL12Rβ1 antibody orprotein of the invention further cross-react with cynomolgous IL12Rβ1polypeptide of SEQ ID NO:98.

In another embodiment, the invention provides antibodies or proteinscomprising the antigen-binding portion thereof, that bind to the sameepitope as do the various specific anti-IL12Rβ1 antibodies mAb1-mAb16 asdescribed herein.

In a certain embodiment, the cross-blocking antibodies or proteins orthe antibody or protein that binds to the same epitope on human IL12Rβ1as any one of mAb1-mAb16, is a human recombinant antibody. Such humanrecombinant antibodies can be prepared and isolated as described in theExamples.

Engineered and Modified Antibodies

Other antibodies or proteins comprising the antigen-binding portionthereof, of the invention can be prepared using an antibody having oneor more of the V_(H) and/or V_(L) sequences of mAb1-mAb16 shown above asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i. e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chainscomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedrecombinant CDR-grafted anti-IL12Rβ1 antibody, comprising the 6 CDRregions of any one of mAb1-mAb16 as defined in Table 4 or 5, yetcontaining different framework sequences from the original antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chainsvariable region genes can be found in the “VBase” human germlinesequence database (available on the Internet atwww.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al., 1992 J. Mol. Biol. 227:776-798; and Cox, J. P.L. et al., 1994 Eur. J Immunol. 24:827-836.

Examples of framework sequences are those that are structurally similarto the framework sequences used in any one of mAb1-mAb16. The V_(H)CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2 and 3 sequences, can begrafted onto framework regions that have the identical sequence as thatfound in the germline immunoglobulin gene from which the frameworksequence derive, or the CDR sequences can be grafted onto frameworkregions that contain one or more mutations as compared to the germlinesequences. For example, it has been found that in certain instances itis beneficial to mutate residues within the framework regions tomaintain or enhance the antigen binding ability of the antibody (seee.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Therefore, in oneembodiment, the invention relates to affinity matured antibodies derivedfrom one of mAb1-mAb16 antibodies. Conservative modifications (asdiscussed above) can be introduced. The mutations may be amino acidsubstitutions, additions or deletions. Moreover, typically no more thanone, two, three, four or five residues within a CDR region are altered.For example, an antibody of the invention is an affinity-maturedantibody comprising the 6 CDRs of one of mAb1-mAb16 and wherein no morethan one, two, three, four or five residues within a CDR region arealtered.

Accordingly, in another embodiment, the invention provides isolatedengineered anti-IL12Rβ1 antibodies comprising a heavy chain variableregion and a light chain variable region which are identical to thecorresponding heavy and light chain variable regions of at least one ofmAb1 to mAb16 antibodies except that the heavy and/or light chain aminoacid sequences of said engineered antibodies contain one, two, three,four or five amino acid substitutions, deletions or additions ascompared to the original sequences.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region of mAb1 to mAb16, which specifically binds to IL12Rβ1.Such frameworks or scaffolds include the 5 main idiotypes of humanimmunoglobulins, or fragments thereof (such as those disclosed elsewhereherein), and include immunoglobulins of other animal species, preferablyhaving humanized aspects. Single heavy-chain antibodies such as thoseidentified in camelids are of particular interest in this regard. Novelframeworks, scaffolds and fragments continue to be discovered anddeveloped by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target protein of SEQ ID NO: 89. Suchcompounds are referred herein as “polypeptides comprising atarget-specific binding region”. Examples of non-immunoglobulinframework are further described in the sections below (camelidantibodies and non-antibody scaffold).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Camelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT Publication No. WO 94/04678.

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. etal., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 BioconjugateChem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineeredlibraries of camelid antibodies and antibody fragments are commerciallyavailable, for example, from Ablynx, Ghent, Belgium. As with otherantibodies of non-human origin, an amino acid sequence of a camelidantibody can be altered recombinantly to obtain a sequence that moreclosely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies tohumans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detecting antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. Patent PublicationNo. 20040161738 published Aug. 19, 2004. These features combined withthe low antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins in bacteriophageand are functional.

Engineered nanobodies can further be customized by genetic engineeringto have a half life in a recipient subject of from 45 minutes to twoweeks. In a specific embodiment, the camelid antibody or nanobody isobtained by grafting the CDRs sequences of the heavy or light chain ofone of the human antibodies of the invention, mAb1 to mAb16, intonanobody or single domain antibody framework sequences, as described forexample in PCT Publication No. WO 94/04678.

Non-Antibody Scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, Adnectins (fibronectin) (Compound Therapeutics, Inc.,Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(i) Fibronectin Scaffold

The fibronectin scaffolds are based preferably on fibronectin type IIIdomain (e.g., the tenth module of the fibronectin type III (10 Fn3domain)). The fibronectin type III domain has 7 or 8 beta strands whichare distributed between two beta sheets, which themselves pack againsteach other to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of one of mAb1 to mAb16 usingstandard cloning techniques.

(ii) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(iii) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, USPatent Publication Nos 20040175756; 20050053973; 20050048512; and20060008844.

(vi) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies; they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(v) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT Publication WO 199916873.

(vi) Affilin—Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368

(vii) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa)mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below.

As used herein, the term “Fc region” is used to define the C-terminalregion of an immunoglobulin heavy chain, including native sequence Fcregion and variant Fc regions. The human IgG heavy chain Fc region isgenerally defined as comprising the amino acid residue from positionC226 or from P230 to the carboxyl-terminus of the IgG antibody. Thenumbering of residues in the Fc region is that of the EU index of Kabat.The C-terminal lysine (residue K447) of the Fc region may be removed,for example, during production or purification of the antibody.Accordingly, a composition of antibodies of the invention may compriseantibody populations with all K447 residues removed, antibodypopulations with no K447 residues removed, and antibody populationshaving a mixture of antibodies with and without the K447 residue.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et at.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret at.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chem 276:6591-6604).

In certain embodiments, the Fc domain of the IgG1 isotype is used. Insome specific embodiments, a mutant variant of the IgG1 Fc fragment isused, e.g. a silent IgG1 Fc which reduces or eliminates the ability ofthe fusion polypeptide to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to bind to an Fcγ receptor. An example of anIgG1 isotype silent mutant is IgG1 wherein Leucineis replaced by Alanineat amino acid positions 234 and 235 as described in J. Virol 2001December; 75(24):12161-8 by Hezareh et al. Another example of an IgG1isotype silent mutant is IgG1 with D265A mutation (aspartate beingsubstituted by alanine at position 265).

In certain embodiments, the Fc domain is a silent Fc mutant preventingglycosylation at position 297 of the Fc domain. For example, the Fcdomain contains an amino acid substitution of asparagine at position297. An example of such amino acid substitution is the replacement ofN297 by a glycine or an alanine.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1 176 195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. Therefore, in one embodiment, theantibodies of the invention are produced by recombinant expression in acell line which exhibits a hypofucosylation pattern, for example, amammalian cell line with deficient expression of the FUT8 gene encodingfucosyltransferase. PCT Publication WO 03/035835 by Presta describes avariant CHO cell line, Lecl3 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).Alternatively, the antibodies of the invention can be produced in ayeast or a filamentous fungus engineered for mammalian-likeglycosylation pattern, and capable of producing antibodies lackingfucose as glycosylation pattern (see for example EP 1 297 172).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.The pegylation can be carried out by an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by theinvention is a conjugate or a protein fusion of at least theantigen-binding region of the antibody of the invention to serumprotein, such as human serum albumin or a fragment thereof to increasehalf-life of the resulting molecule. Such approach is for exampledescribed in Ballance et al. EP 0 322 094.

Another possibility is a fusion of at least the antigen-binding regionof the antibody of the invention to proteins capable of binding to serumproteins, such human serum albumin to increase half life of theresulting molecule. Such approach is for example described in Nygren etal., EP 0 486 525.

Methods of Engineering Altered Antibodies

As discussed above, the anti-IL12Rβ1 antibodies having V_(H) and V_(L)sequences or full length heavy and light chain sequences shown hereincan be used to create new anti-IL12Rβ1 antibodies by modifying fulllength heavy chain and/or light chain sequences, V_(H) and/or V_(L)sequences, or the constant region(s) attached thereto. Thus, in anotheraspect of the invention, the structural features of an anti-IL12Rβ1antibody of the invention are used to create structurally relatedanti-IL12Rβ1 antibodies that retain at least one functional property ofthe antibodies of the invention, such as binding to human IL12Rβ1 andalso inhibiting one or more functional properties of IL12Rβ1 (e.g.,inhibiting IL12 and/or IL23 binding to IL12Rβ1, inhibiting IL12 inducedIFNγ production in blood cells, etc).

For example, one or more CDR regions of any one of mAb1 to mAb16, ormutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-IL12Rβ1 antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences of mAb1-mAb16provided in the tables above, or one or more CDR region thereof. Tocreate the engineered antibody, it is not necessary to actually prepare(i.e., express as a protein) an antibody having one or more of the V_(H)and/or V_(L) sequences of mAb1 or mAb16, or one or more CDR regionsthereof. Rather, the information contained in the sequence(s) is used asthe starting material to create a “second generation” sequence(s)derived from the original sequence(s) and then the “second generation”sequence(s) is prepared and expressed as a protein.

The second generation sequences are derived for example by altering theDNA coding sequence of at least one amino acid residue within the heavychain variable region antibody sequence and/or the light chain variableregion antibody sequence of any one of mAb1 to mAb16, to create at leastone altered antibody sequence; and expressing the altered antibodysequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IL12Rβ1 antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence a full length light chain antibody sequence of any one of mAb1to mAb16; altering at least one codon in the nucleotide coding sequence,said codon encoding an amino acid residue within the full length heavychain antibody sequence and/or the full length light chain antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

The altered antibody sequence can also be prepared by screening antibodylibraries having unique heavy and light CDR3 sequences of any one ofmAb1-mAb16 respectively, or minimal essential binding determinants asdescribed in US Patent Publication No. 20050255552, and alternativesequences for CDR1 and CDR2 sequences. The screening can be performedaccording to any screening technology appropriate for screeningantibodies from antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of the desiredfunctional properties of the anti-IL12Rβ1 antibodies described herein,which functional properties include, but are not limited to,specifically binding to human IL12Rβ1; and/or it inhibits IL12 and IL23binding to IL12Rβ1 polypeptide; and/or it inhibits IL12 induced IFNγproduction in human blood cells; it inhibits IL23 induced IFNγproduction in human blood cells; and/or it inhibits IL12 ex vivo IFN-γproduction in primate blood cells.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-IL12Rβ1 antibody coding sequence and the resultingmodified anti-IL12Rβ1 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies or proteins of the invention. Examples of variablelight chain nucleotide sequences are those encoding the variable lightchain amino acid sequences of any one of mAb1 to mAb16, the lattersequences being derived from the Table 3 (showing the entire nucleotidecoding sequences of heavy and light chains of mAb1 to mAb16) and Table 2(showing the amino acid sequences of the variable regions of mAb1 tomAb16.

The invention also pertains to nucleic acid molecules that derive fromthe latter sequences having been optimized for protein expression inmammalian cells, for example, CHO cell lines.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. Once DNA fragments encoding, for example, V_(H) andV_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to an scFv gene. In these manipulations, aV_(L)- or V_(H)-encoding DNA fragment is operatively linked to anotherDNA molecule, or to a fragment encoding another protein, such as anantibody constant region or a flexible linker. The term “operativelylinked”, as used in this context, is intended to mean that the two DNAfragments are joined in a functional manner, for example, such that theamino acid sequences encoded by the two DNA fragments remain in-frame,or such that the protein is expressed under control of a desiredpromoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., el al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments,the heavy chain constant region is selected among IgG1 isotypes. For aFab fragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., 1988 Science 242:423-426; Huston et at., 1988 Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

Isolation of Recombinant Antibodies of the Invention

A variety of methods of screening antibodies have been described in theArt. Such methods may be divided into in vivo systems, such astransgenic mice capable of producing fully human antibodies upon antigenimmunization and in vitro systems, consisting of generating antibody DNAcoding libraries, expressing the DNA library in an appropriate systemfor antibody production, selecting the clone that express antibodycandidate that binds to the target with the affinity selection criteriaand recovering the corresponding coding sequence of the selected clone.These in vitro technologies are known as display technologies, andinclude without limitation, phage display, RNA or DNA display, ribosomedisplay, yeast or mammalian cell display. They have been well describedin the Art (for a review see for example: Nelson et al., 2010 NatureReviews Drug discovery, “Development trends for human monoclonalantibody therapeutics” (Advance Online Publication) and Hoogenboom etal. in Method in Molecular Biology 178:1-37, O'Brien et al., ed., HumanPress, Totowa, N.J., 2001). In one specific embodiment, humanrecombinant antibodies of the invention are isolated using phage displaymethods for screening libraries of human recombinant antibody libraries,such as HuCAL® libraries.

Repertoires of V_(H) and V_(L) genes or related CDR regions can beseparately cloned by polymerase chain reaction (PCR) or synthesized byDNA synthesizer and recombined randomly in phage libraries, which canthen be screened for antigen-binding clones. Such phage display methodsfor isolating human antibodies are established in the art or describedin the examples below. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

In a certain embodiment, human antibodies directed against IL12Rβ1 canbe identified using transgenic or transchromosomic mice carrying partsof the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous p and K chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et at., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL12Rβ1 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL12Rβ1 antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Monoclonal Antibodies of the Invention from the MurineSystem

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific or epitope-specific antibodies. Forexample, single cell suspensions of splenic lymphocytes from immunizedmice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecretingmouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated atapproximately 2×145 in flat bottom microtiter plates, followed by a twoweek incubation in selective medium containing 20% fetal Clone Serum,18% “653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mMsodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/mlpenicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma;the HAT is added 24 hours after the fusion). After approximately twoweeks, cells can be cultured in medium in which the HAT is replaced withHT. Individual wells can then be screened by ELISA for human monoclonalIgM and IgG antibodies. Once extensive hybridoma growth occurs, mediumcan be observed usually after 10-14 days. The antibody secretinghybridomas can be replated, screened again, and if still positive forhuman IgG, the monoclonal antibodies can be subcloned at least twice bylimiting dilution. The stable subclones can then be cultured in vitro togenerate small amounts of antibody in tissue culture medium forcharacterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention can be produced in a host cell transfectomausing, for example, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (e.g., Morrison, S.(1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains can beobtained by standard molecular biology or biochemistry techniques (e.g.,DNA chemical synthesis, PCR amplification or cDNA cloning using ahybridoma that expresses the antibody of interest) and the DNAs can beinserted into expression vectors such that the genes are operativelylinked to transcriptional and translational control sequences. In thiscontext, the term “operatively linked” is intended to mean that anantibody gene is ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the antibodygene. The expression vector and expression control sequences are chosento be compatible with the expression host cell used. The antibody lightchain gene and the antibody heavy chain gene can be inserted intoseparate vector or, more typically, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present). The light and heavy chainvariable regions of the antibodies described herein can be used tocreate full-length antibody genes of any antibody isotype by insertingthem into expression vectors already encoding heavy chain constant andlight chain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, forexample mammalian host cells, yeast or filamentous fungi, is discussedbecause such eukaryotic cells, and in particular mammalian cells, aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody.

In one specific embodiment, a cloning or expression vector according tothe invention comprises either at least one of the following codingsequences (a)-(p) operatively linked to suitable promoter sequencesselected from the group consisting of:

(a) SEQ ID NO:73 and SEQ ID NO:85;

(b) SEQ ID NO:77 and SEQ ID NO:85;

(c) SEQ ID NO:81 and SEQ ID NO:85;

(d) SEQ ID NO:74 and SEQ ID NO:86;

(e) SEQ ID NO:78 and SEQ ID NO:86;

(f) SEQ ID NO:82 and SEQ ID NO:86;

(g) SEQ ID NO:75 and SEQ ID NO:87;

(h) SEQ ID NO:79 and SEQ ID NO:87;

(i) SEQ ID NO:83 and SEQ ID NO:87;

(j) SEQ ID NO:76 and SEQ ID NO:88;

(k) SEQ ID NO:80 and SEQ ID NO:88;

(l) SEQ ID NO:84 and SEQ ID NO:88;

(m) SEQ ID NO:94 and SEQ ID NO:85;

(n) SEQ ID NO:95 and SEQ ID NO:86;

(o) SEQ ID NO:96 and SEQ ID NO:87; and,

(p) SEQ ID NO:97 and SEQ ID NO:88.

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621), CHOK1 dhfr+cell lines, NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another expression system is the GS geneexpression system shown in PCT Publications WO 87/04462, WO 89/01036 andEP 0 338 841. In one embodiment, mammalian host cells for expressing therecombinant antibodies of the invention include mammalian cell linesdeficient for FUT8 gene expression, for example as described in U.S.Pat. No. 6,946,292.

When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods (See for example Abhinav et al. 2007,Journal of Chromatography 848: 28-37).

In one specific embodiment, the host cell of the invention is a hostcell transfected with an expression vector having the coding sequencesselected from the group consisting of (a)-(p) suitable for theexpression of mAb1-mAb16 respectively, operatively linked to suitablepromoter sequences:

(a) SEQ ID NO:73 and SEQ ID NO:85;

(b) SEQ ID NO:77 and SEQ ID NO:85;

(c) SEQ ID NO:81 and SEQ ID NO:85;

(d) SEQ ID NO:74 and SEQ ID NO:86;

(e) SEQ ID NO:78 and SEQ ID NO:86;

(f) SEQ ID NO:82 and SEQ ID NO:86;

(g) SEQ ID NO:75 and SEQ ID NO:87;

(h) SEQ ID NO:79 and SEQ ID NO:87;

(i) SEQ ID NO:83 and SEQ ID NO:87;

(j) SEQ ID NO:76 and SEQ ID NO:88;

(k) SEQ ID NO:80 and SEQ ID NO:88;

(l) SEQ ID NO:84 and SEQ ID NO:88;

(m) SEQ ID NO:94 and SEQ ID NO:85;

(n) SEQ ID NO:95 and SEQ ID NO:86;

(o) SEQ ID NO:96 and SEQ ID NO:87; and,

(p) SEQ ID NO:97 and SEQ ID NO:88.

The latter host cells may then be further cultured under suitableconditions for the expression and production of an antibody of theinvention selected from the group consisting of mAb1-mAb16 respectively.

Immunoconjugates

In another aspect, the present invention features an anti-IL12Rβ1antibody of the invention, or a fragment thereof, conjugated to atherapeutic moiety, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Such conjugates are referred toherein as “immunoconjugates”. Immunoconjugates that include one or morecytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells.Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thioepachloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody of the invention include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg™;Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.,2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al., 2003 CancerImmunol. Immunother. 52:328-337; Payne, G., 2003 Cancer Cell 3:207-212;Allen, T. M., 2002 Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman,R. J., 2002 Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P. D. andSpringer, C. J., 2001 Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰, and lutetium¹⁷⁷. Method for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (DEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL1”), interleukin-2(“IL2”), interleukin-6 (“IL6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et at., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Inmunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific ormultispecific molecules comprising an anti-IL12Rβ1 antibody of theinvention. An antibody of the invention can be derivatized or linked toanother functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. The antibody of the invention may in fact be derivatized orlinked to more than one other functional molecule to generatemulti-specific molecules that bind to more than two different bindingsites and/or target molecules; such multi-specific molecules are alsointended to be encompassed by the term “bispecific molecule” as usedherein. To create a bispecific molecule of the invention, an antibody ofthe invention can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother binding molecules, such as another antibody, antibody fragment,peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for IL12Rβ1, forexample, one antigen-binding portion of any one of mAb1-mAb16 and asecond binding specificity for a second target epitope. For example, thesecond target epitope is another epitope of IL12Rβ1 different from thefirst target epitope. Another example is a bispecific moleculecomprising at least one first binding specificity for IL12Rβ1, forexample, one antigen-binding portion of any one of mAb1-mAb16 and asecond binding specificity for an epitope within IL12Rβ2 or IL23Rα.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding-specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particular embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present invention provides multivalent antibodiescomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to IL12Rβ1, for example,selected from antigen-binding portions of any one of mAb1-mAb16. In oneembodiment, the multivalent antibodies provide at least two, three orfour antigen-binding portions of the antibodies. The antigen-bindingportions can be linked together via protein fusion or covalent or noncovalent linkage. Alternatively, methods of linkage have been describedfor the bispecific molecules. Tetravalent compounds can be obtained forexample by cross-linking antibodies of the antibodies of the inventionwith an antibody that binds to the constant regions of the antibodies ofthe invention, for example the Fc or hinge region.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofantibodies of the present invention, for example, one antibody selectedfrom the group consisting of mAb1-mAb16, formulated together with apharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) antibodies, orimmunoconjugates or bispecific molecules of the invention. For example,a pharmaceutical composition of the invention can comprise a combinationof antibodies that bind to different epitopes on the target antigen orthat have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-IL12Rβ1 antibody of the presentinvention, for example one antibody selected from the group consistingof mAb1-mAb16, combined with at least one other anti-inflammatory oranother chemotherapeutic agent, for example, an immunosuppressant agent.Examples of therapeutic agents that can be used in combination therapyare described in greater detail below in the section on uses of theantibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). In oneembodiment, the carrier should be suitable for subcutaneous route.Depending on the route of administration, the active compound, i.e.,antibody, immunoconjugate, or bispecific molecule, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The pharmaceutical compositions of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Reviews on the development of stable protein (e;g; antibody)formulations may be found in Cleland et al. (1993) Crit. Reviews. Ther.Drug Carrier Systems 10(4):307-377 and Wei Wang (1999) Int. J.Pharmaceutcs 185:129-88. Additional formulation discussions forantibodies may be found, e.g., in Daugherty and Mrsny (2006) AdvancedDrug Delivery Reviews 58: 686-706; U.S. Pat. Nos. 6,171,586, 4,618,486,US Publication No. 20060286103, PCT Publication WO 06/044908, WO07/095337, WO 04/016286, Colandene et al. (2007) J. Pharm. Sci 96:1598-1608; Schulman (2001) Am. J. Respir. Crit. Care Med. 164:S6-S11 andother known references.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents, antibacterial agents such as benzyl alcohol ormethyl parabens, antioxidants such as ascorbic acid or sodium bisulfite,chelating agents such ethylenediaminetetraacetic acid, buffers such asacetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposables syringes ormultiple dose vials made of glass or plastic.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the antibodies or proteins of the invention into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 per centto about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 per cent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Dosage regimens for an anti-IL12Rβ1 antibody orprotein of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight by intravenous administration, with the antibody being givenusing one of the following dosing schedules: every four weeks for sixdosages, then every three months; every three weeks; 3 mg/kg body weightonce followed by 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibodies or proteins of the invention are usually administered onmultiple occasions. Intervals between single dosages can be, forexample, weekly, monthly, every three months or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody tothe target antigen in the patient. In some methods, dosage is adjustedto achieve a plasma antibody concentration of about 1-1000 μg/ml and insome methods about 25-300 μg/ml.

Alternatively, antibody or protein can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf-life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-IL12Rβ1 antibody orprotein of the invention can result in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction.

A composition of the present invention can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the inventioninclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion.

The phrase “parenteral administration” as used herein means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrastemal injection and infusion.

Alternatively, an antibody or protein of the invention can beadministered by a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The antibodies or proteins of the invention can be prepared withcarriers that will protect the antibodies against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. Many other such implants, deliverysystems, and modules are known to those skilled in the art.

In certain embodiments, the antibodies or proteins of the invention canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade,1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M.Owais et al., 1995 Antimicrob. Agents Chernother. 39:180); surfactantprotein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion; I. J. Fidler,1994 Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies or proteins of the present invention have in vitro and invivo diagnostic and therapeutic utilities. For example, these moleculescan be administered to cells in culture, e.g. in vitro or in vivo, or ina subject, e.g., in vivo, to treat, prevent or diagnose a variety ofdisorders.

The methods are particularly suitable for treating, preventing ordiagnosing IL12Rβ1-related disorders and/or autoimmune and inflammatorydisorders, e.g., rheumatoid arthritis, psoriasis or inflammatory boweldiseases.

The invention also provides methods for decreasing or suppressing IL12or IL23 induced signaling response in human blood cells by administeringa composition comprising a therapeutically efficient dose of theantibodies of the invention.

As used herein, an “IL12Rβ1-related disorder” includes conditionsassociated with or characterized by aberrant IL12 and/or IL23 levelsand/or diseases or conditions that can be treated by reducing orsuppressing IL12 and/or IL23 induced signaling activity in human bloodcells e.g. the production of IFNγ or IL17 as measured in plasma or theextent of phosphorylation of STAT4 protein as measured byflow-cytometric methods or western blot. These include inflammatoryconditions and autoimmune diseases, such as rheumatoid arthritis,psoriasis and inflammatory bowel diseases. These further includeallergies and allergic conditions, hypersensitivity reactions, and organor tissue transplant rejection.

For example, the antibodies or proteins of the invention may be used forthe treatment of recipients of heart, lung, combined heart-lung, liver,kidney, pancreatic, skin or corneal transplants, including allograftrejection or xenograft rejection, and for the prevention ofgraft-versus-host disease, such as following bone marrow transplant, andorgan transplant associated arteriosclerosis.

The antibodies or proteins of the invention are useful for thetreatment, prevention, or amelioration of autoimmune disease and ofinflammatory conditions, in particular inflammatory conditions with anaetiology including an autoimmune component such as arthritis (forexample rheumatoid arthritis, arthritis chronica progrediente andarthritis deformans) and rheumatic diseases, including inflammatoryconditions and rheumatic diseases involving bone loss, inflammatorypain, spondyloarhropathies including ankolsing spondylitis, Reitersyndrome, reactive arthritis, psoriatic arthritis, and enterophathisarthritis, hypersensitivity (including both airways hypersensitivity anddermal hypersensitivity) and allergies. Specific auto-immune diseasesfor which antibodies of the invention may be employed include autoimmunehaematological disorders (including e.g. hemolytic anaemia, aplasticanaemia, pure red cell anaemia and idiopathic thrombocytopenia),systemic lupus erythematosus, inflammatory muscle disorders,polychondritis, sclerodoma, Wegener granulomatosis, dermatomyositis,chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnsonsyndrome, idiopathic sprue, autoimmune inflammatory bowel disease(including e.g. ulcerative colitis, Crohn's disease and Irritable BowelSyndrome), endocrine ophthalmopathy, Graves disease, sarcoidosis,multiple sclerosis, primary biliary cirrhosis, juvenile diabetes(diabetes mellitus type I), uveitis (anterior and posterior),keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, psoriatic arthritis and glomerulonephritis (with andwithout nephrotic syndrome, e.g. including idiopathic nephrotic syndromeor minimal change nephropathy), tumors, multiple sclerosis, inflammatorydisease of skin and cornea, myositis, loosening of bone implants,metabolic disorders, such as atherosclerosis, diabetes, anddislipidemia.

The antibodies or proteins of the invention may also be useful for thetreatment, prevention, or amelioration of asthma, bronchitis,pneumoconiosis, pulmonary emphysema, and other obstructive orinflammatory diseases of the airways.

The antibodies or proteins of the invention may also be useful fortreating diseases of bone metabolism including osteoarthritis,osteoporosis and other inflammatory arthritides, and bone loss ingeneral, including age-related bone loss, and in particular periodontaldisease.

The antibodies or proteins of the invention may be administered as thesole active ingredient or in conjunction with, e.g. as an adjuvant to orin combination to, other drugs e.g. immunosuppressive orimmunomodulating agents or other anti-inflammatory agents or othercytotoxic or anti-cancer agents, e.g. for the treatment or prevention ofdiseases mentioned above. For example, the antibodies of the inventionmay be used in combination with DMARD, e.g. Gold salts, sulphasalazine,antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolicacid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide,glucocorticoids; a calcineurin inhibitor, e.g. cyclosporin A or FK 506;a modulator of lymphocyte recirculation, e.g. FTY720 and FTY720 analogs;a mTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin,CC1779, ABT578, AP23573 or TAFA-93; an ascomycin havingimmuno-suppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclo-phos-phamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid; myco-pheno-late mofetil;15-deoxyspergualine or an immunosuppressive homologue, analogue orderivative thereof; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands;other immunomodulatory compounds, e.g. a recombinant binding moleculehaving at least a portion of the extracellular domain of CTLA4 or amutant thereof, e.g. an at least extracellular portion of CTLA4 or amutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig (forex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesionmolecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists,VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent,e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or5-fluorouracil; anti TNF agents, e.g. monoclonal antibodies to TNF, e.g.infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI orTNF-RII, e.g. Etanercept, PEG-TNF-RI; blockers of proinflammatorycytokines, IL1 blockers, e.g. Anakinra or IL1 trap, AAL160, IL17blockers, IL13 blockers, IL4 blockers, IL6 blockers; chemokinesblockers, e.g inhibitors or activators of proteases, e.g.metalloproteases, anti-IL15 antibodies, anti-IL6 antibodies, anti-IL17antibodies, anti-IL4 antibodies, anti-IL13 antibodies, anti-CD20antibodies, anti-Blys or anti-BAFFR antibodies, NSAIDs, such as aspirinor an anti-infectious agent (list not limited to the agent mentioned).

In accordance with the foregoing the present invention provides in a yetfurther aspect:

A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount ofan anti-IL12Rβ1 antibody or protein of the invention, and at least onesecond drug substance, said second drug substance being aimmuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeuticor anti-infectious drug, e.g. as indicated above.

Or, a therapeutic combination, e.g. a kit, comprising of atherapeutically effective amount of a) an antibody or protein of theinvention, and b) at least one second substance selected from aimmuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeuticor anti-infectious drug, e.g. as indicated above. The kit may compriseinstructions for its administration.

Where the antibodies of the invention are administered in conjunctionwith other immuno-suppressive/immunomodulatory, anti-inflammatorychemotherapeutic or anti-infectious therapy, dosages of theco-administered combination compound will of course vary depending onthe type of co-drug employed, e.g. whether it is a DMARD, anti-TNF, IL1blocker or others, on the specific drug employed, on the condition beingtreated and so forth.

In one specific embodiment, the antibodies of the invention may beadministered in combination with anti TNF agents.

In another embodiment, the antibodies of the invention are administeredonly to patient population which is selected among patients sufferingfrom systemic lupus eryhematous or rheumatoid arthritis and exhibitingan abnormal serum level of IL12 respectively IFNγ or IL17 or elevatedlevels and frequency of phosphoSTAT4 in blood cells. In otherembodiment, the antibodies of the invention are administered only topatient population which is selected among group of patients whichrespond to anti-IL12 or anti-p40 treatment. Biomarkers that identifypatients that have an increased likelihood of responding to anti-IL12(or anti-p40) treatment may be any of the following without beinglimited to these: elevated levels of serum IL12, elevated levels ofcertain T cell subsets, mRNA levels of IFNγ, TNFα, IL12Rβ2 or STAT4 fromisolated peripheral blood mononuclear cells (PBMCs), phosphoSTAT4expression in skin biopsies respectively PBMCs.

In one embodiment, the antibodies or proteins of the invention can beused to detect levels of IL12Rβ1, or levels of cells that containIL12Rβ1. This can be achieved, for example, by contacting a sample (suchas an in vitro sample) and a control sample with the anti-IL12Rβ1antibody under conditions that allow for the formation of a complexbetween the antibody and IL12Rβ1. Any complexes formed between theantibody and IL12Rβ1 are detected and compared in the sample and thecontrol. For example, standard detection methods, well known in the art,such as ELISA and flow cytometic assays, can be performed using thecompositions of the invention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of IL12Rβ1 (e.g., human IL12Rβ1 antigen) in asample, or measuring the amount of IL12Rβ1, comprising contacting thesample, and a control sample, with an antibody or protein of theinvention, or an antigen binding region thereof, which specificallybinds to IL12Rβ1, under conditions that allow for formation of a complexbetween the antibody or portion thereof and IL12Rβ1. The formation of acomplex is then detected, wherein a difference in complex formationbetween the sample compared to the control sample is indicative of thepresence of IL12Rβ1 in the sample.

Also within the scope of the invention are kits consisting of thecompositions (e.g., antibodies, proteins, human antibodies andbispecific molecules) of the invention and instructions for use. The kitcan further contain a least one additional reagent, or one or moreadditional antibodies or proteins of the invention (e.g., an antibodyhaving a complementary activity which binds to an epitope on the targetantigen distinct from the first antibody). Kits typically include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit. The kit may furthercomprise tools for diagnosing whether a patient belongs to a group thatwill respond to an anti-IL12Rβ1 antibody treatment, as defined above.

The invention having been fully described is now further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting.

EXAMPLES

Methods

1. Affinity Determination Using Surface Plasmon Resonance (BiacoreSystem)

For determination of K_(D) values, surface plasmon resonance technologywas applied using the Biacore™ technology. Anti-human-Fc-capture CM5chip (Biacore, Sweden) was used for capturing IL12Rβ1-Fc-Fusion followedby ligand (Fab) injection at different concentrations.

2. IL 12Rβ1-IL12/IL23 In Vitro Competitive Binding Inhibition Assay(Bioveris)

The IC₅₀ represents the concentration of a Fab or IgG that was requiredfor 50% inhibition of a receptor/ligand interaction. During the assayprocedure, a fixed amount (predetermined to result in near maximumsignal) of the receptor was incubated with increasing concentrations(1:100 to 100:1 molar ratio) of purified Fab/IgG in solution.Subsequently, the samples were transferred to a ligand coated andblocked MSD multi-titer plate. The free receptor binds to the ligand andwas detected via an appropriate ruthenium complex-labeled antibody andquantified. The concentration of bound receptor as an inverse functionof Fab/IgG concentration used for incubation was fitted using therespective model. The inflection point of the fitted curve representsthe IC₅₀ value.

3. Inhibition of IL12-dependent IFNγ production of human blood cells

Peripheral blood mononuclear cells (PBMCs) from donor blood wereisolated via commonly used Ficoll/Histopaque gradient (Noble and Cutts,1967 CanVetJ 8(5): 110-111). Cells were adjusted to 2E+6 cells/ml (inX-Vivo 15 medium). 50 μl cells (1E+5) were transferred to a 96 well Ubottom plate and incubated with inhibitory antibodies, e.g anti humanIL12Rβ1 Fabs or silent IgG1 or control mAbs or controls at desiredconcentrations and pre-incubated for 30 min at RT on a shaker.Stimulation with 2 μg/ml anti-CD3 and anti-CD28 mAbs and 2 ng/mlrecombinant human cytokine IL12 was performed o/n, for 20 hrs at 37° C.in a 5% CO2 incubator. Next day, the supernatant was collected bycentrifugation of the cells at 250g for 5 min at RT and transferred to afresh 96 well plate and used for ELISA determination or stored at −20°C. until assay was performed.

For the IFNγ ELISA the above collected supernatants were diluted inX-Vivo15 medium and the ELISA was performed according manufacturesprotocols BenderMed Systems #BMS228HS or Biozol/Biolegend #BLD-430105.IFNγ production was determined according to IFNγ standard titrationcurve.

4. Inhibition of IL23-Dependent IFNγ Production of Human Blood Cells

Another assay system was investigated, using PHA-stimulated PBMC. Inthis cell population, the T cells proliferate upon lectin exposure andthus the proportion of T cells in the population increases. Inpreliminary experiments, the responsiveness of these cells to IL-12,IL-23, IL-18 and LPS, alone or in combination was evaluated and theoptimal stimulation conditions were established. The effects ofIL-12+IL-18 and IL-23+IL-18 on IFN-γ secretion were induction of around7 ng/ml and 800 pg/ml, respectively.

Alternatively, PBMCs from human donor blood were isolated bycentrifugation over a density gradient and incubated with serialdilutions of IgGs for 30 min at RT. IL-18 and IL-23 were added and cellswere incubated for 48 h at 37° C./5%/CO₂. 5-fold diluted supernatants(in PBS/2 mM EDTA to achieve concentrations within the range of thestandard curve 10 ng/ml-20 pg/ml) were taken to quantify IFN-γ bystandard sandwich ELISA.

5. Inhibition of IL 12-Dependent IFNγ Production in Whole Blood

Aliquots of anti-coagulated blood of human or cynomolgous origin weredistributed to individual wells of U-bottom 96 well plates (Costar,3799) and serial dilutions of mAbs in X-Vivo 15 medium were added andincubated for 30 min at RT. After adding the human cytokines IL-12 andIL-18 the cells were incubated for 20-24 h at 37° C., 5% CO₂, beforesampling the supernatants to quantify IFN-γ by ELISA.

6. ADCC Assay

In contrast to calcein, its acetoxy-methyl ester (Calcein-AM) is acompound able to pass cell membranes. Within the cell the ester bondsare hydrolyzed by cellular esterases. Thus the fluorescent calcein istrapped inside the cell. It will leak out only if membrane integrity isdisturbed, i.e. if a cell is killed. Calcein-labelled target cells serveas targets for the NK effector cells, and the amount of calcein measuredin the supernatant is proportional to the ability of a antibodiespreparation to mediate ADCC.

For the NK3.3 assay it is necessary to starve the NK3.3 cells byculturing them in starvation medium (without IL-2 and IL-10) for twodays.

Human IL-12Rβ1 transfected murine pre-B-cells were labelled with calceinand incubated with the antibodies to be tested. The human B cell lineRaji expressing CD20 and anti-CD20 mAbs are used as controls for thekilling activity of the NK3.3 cells. Spontaneous release from labelledcells only and background killing by the addition of NK3.3 cells withoutmAbs are also determined. In correlation with total release induced bycell lysis with Triton-X100 the % specific lysis is determined.

7. Cynomolgus Monkey pharmacodynamics (PD) assay

Heparinized blood samples were distributed in 96-U well plates (1090μl/well). Recombinant human IL-12 (R&D Systems; 100 ng/ml final) andIL-18 (MBL; 50 ng/ml final) were added to each well and the plates weremixed gently for 3 minutes. After an incubation of 24 hrs at 37° C., in6% CO₂, the plates were centrifuged at 2000 rpm for 10 min. The plasmawere collected and kept at −80° C. until further processing.

IL-2, TNFα and IFN-γ determination was performed with NHP specificELISA-kits (CT711, CT148 and CT141), as described by the manufacturer(UcyTech Biosciences, Utrecht).

For the PD readout, the results in pg of INFγ/ml were corrected by thenumber of lymphocytes found in each sample to be finally expressed aspg/10⁶ lymphocyte.

8. Rat In Vivo Compatibility Assay

Rats are injected with defined doses of mAbs and blood samples taken atseveral intervals to monitor the peak plasma concentration and the rateof elimination to determine the plasma half life time. Since nocross-reactivity to the rat target is expected also no target-relatedeffects (internalization, turnover) can be expected to influenceresults.

9. CD45RBhi Transfer Inflammatory Bowel Disease Mouse Model

To elicit the disease characterized by weight loss CD4+CD45RBHi Tlymphocytes are isolated from BALB/c mouse spleens by FACS-sorting andinjected (2×10⁵ cells/mouse, i.p.) into 10 week old female SCID mice(day 0). Negative control mice receive PBS i.p. Groups of mice receivetreatment by subcutaneous or intraperitoneal injection of mAbs(anti-IL12p40 clone C17.8 or anti-IL12Rβ1 antibody or isotype control)or PBS on d1, 7, 14 and 21. The body weight of each mouse is monitoredthroughout and at the end of the study. Histological examination of theterminal colon and determination of serum haptoglobin are determined atnecropsy on d28.

10. Biacore Cross-Blocking Assay

The following generally describes a suitable Biacore assay fordetermining whether an antibody or other binding agent cross-blocks orwas capable of cross-blocking antibodies according to the invention. Itwill be appreciated that the assay can be used with any of the IL12Rβ1binding agents described herein.

The Biacore machine (for example the BIAcore 3000) is operated in linewith the manufacturer's recommendations.

IL12Rβ1 extracellular domain may be coupled to e.g. a CM5 Biacore chipby way of routinely used amine coupling chemistry, e.g. EDC-NHS aminecoupling, to create a IL12Rβ1-coated surface. In order to obtainmeasurable levels of binding, typically 200-800 resonance units ofIL12Rβ1 may be coupled to the chip (this amount gives measurable levelsof binding and is at the same time readily saturable by theconcentrations of test reagent being used).

An alternative way of attaching IL12Rβ1 to the BIAcore chip is by usinga “tagged” version of IL12Rβ1, for example N-terminal or C-terminalHis-tagged IL12Rβ1. In this format, an anti-His antibody would becoupled to the Biacore chip and then the His-tagged IL12Rβ1 would bepassed over the surface of the chip and captured by the anti-Hisantibody.

The two antibodies to be assessed for their ability to cross-block eachother are mixed in a stoechiometrical amount, e.g. at a one to one molarratio, of binding sites in a suitable buffer to create the test mixture.The buffer used is typically a buffer which is normally used in proteinchemistry, such as e.g. PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄,1.76 mM KH₂PO₄, pH7.4). When calculating the concentrations on a bindingsite-basis the molecular weight of an antibody is assumed to be thetotal molecular weight of the antibody divided by the number of target(i.e. IL12Rβ1) binding sites on that antibody.

The concentration of each antibody in the test mixture should be highenough to ensure saturation of the binding sites for that antibody onthe IL12Rβ1 molecule which are bound on the BIAcore chip. The antibodiesin the mixture are at the same molar concentration (on a binding basis)and that concentration would typically be between 1.0 mM and 1.5 mM (ona binding site basis).

Separate solutions containing the separate antibodies on their own arealso prepared. The buffer used for these separate solutions should bethe same buffer and at the same concentration as is used for the testmixture.

The test mixture is passed over the IL12Rβ1-coated BIAcore chip and thebinding recorded. The bound antibodies were thereafter removed bytreating the chip with e.g. an acid, such as 30 mM HCl for about 1minute. It is important that the IL12Rβ1 molecules which are bound tothe chip are not damaged.

The solution of the first antibody alone is then passed over theIL12Rβ1-coated surface and the binding was recorded. Thereafter, thechip is treated to remove all of the bound antibody without damaging thechip-bound IL12Rβ1, e.g. by way of above mentioned acid treatment.

The solution of the second antibody alone is then passed over theIL12Rβ1-coated surface and the amount of binding recorded.

The maximal theoretical binding can be defined as the sum of the bindingto IL12Rβ1 of each antibody separately. This is then compared to theactual binding of the mixture of antibodies measured. If the actualbinding is lower than that of the theoretical binding, the twoantibodies are cross-blocking each other.

11. ELISA-Based Cross-Blocking Assay

Cross-blocking of an anti-IL12Rβ1 antibody or another IL12Rβ1 bindingagent may also be detected by using an ELISA assay.

The general principle of the ELISA-assay involves coating ananti-IL12Rβ1 antibody onto the wells of an ELISA plate. An excess amountof a second, potentially cross-blocking, anti-IL12Rβ1 antibody is thenadded in solution (i.e. not bound to the ELISA plate). A limited amountof IL12Rβ1-Fc is then added to the wells.

The antibody which is coated onto the wells and the antibody in solutionwill compete for binding of the limited number of IL12Rβ1 molecules. Theplate is then washed to remove IL12Rβ1-Fc that has not bound to thecoated antibody and to also remove the second, solution phase, antibodyas well as any complexes formed between the second, solution phaseantibody and IL12Rβ1-Fc. The amount of bound IL12Rβ1 is then measuredusing an appropriate IL12Rβ1 detection reagent. An antibody in solutionthat is able to cross-block the coated antibody will be able to cause adecrease in the number of IL12Rβ1 molecules that the coated antibody canbind relative to the number of IL12Rβ1 molecules that the coatedantibody can bind in the absence of the second, solution phase,antibody.

This assay is described in more detail further below for two antibodiestermed Ab-X and Ab-Y. In the instance where Ab-X is chosen to be theimmobilized antibody, it is coated onto the wells of the ELISA plate,after which the plates are blocked with a suitable blocking solution tominimize non-specific binding of reagents that are subsequently added.An excess amount of Ab-Y is then added to the ELISA plate such that themoles of Ab-Y IL12Rβ1 binding sites per well are at least 10 fold higherthan the moles of Ab-X IL12Rβ1 binding sites that are used, per well,during the coating of the ELISA plate. IL12Rβ1-Fc is then added suchthat the moles of IL12Rβ1-Fc added per well were at least 25-fold lowerthan the moles of Ab-X IL12Rβ1 binding sites that are used for coatingeach well. Following a suitable incubation period, the ELISA plate iswashed and a IL12Rβ1 detection reagent is added to measure the amount ofIL12Rβ1 specifically bound by the coated anti-IL12Rβ1 antibody (in thiscase Ab-X). The background signal for the assay is defined as the signalobtained in wells with the coated antibody (in this case Ab-X), secondsolution phase antibody (in this case Ab-Y), buffer only (i.e. noIL12Rβ1) and IL12Rβ1 detection reagents. The positive control signal forthe assay is defined as the signal obtained in wells with the coatedantibody (in this case Ab-X), second solution phase antibody buffer only(i.e. no second solution phase antibody), IL12Rβ1 and IL12Rβ1 detectionreagents. The ELISA assay needs to be run in such a manner so as to havethe positive control signal be at least 6 times the background signal.

To avoid any artifacts (e.g. significantly different affinities betweenAb-X and Ab-Y for IL12Rβ1) resulting from the choice of which antibodyto use as the coating antibody and which to use as the second(competitor) antibody, the cross-blocking assay needs to be run in twoformats: 1) format 1 is where Ab-X is the antibody that is coated ontothe ELISA plate and Ab-Y is the competitor antibody that is in solutionand 2) format 2 is where Ab-Y is the antibody that is coated onto theELISA plate and Ab-X is the competitor antibody that is in solution.

12. Cell Binding Assays

Cell binding of selected antibodies is tested using various IL-12Rβ1expressing cell lines, e.g. Kit 225 or HSC-F, or human and cyno T cellblasts, employing flow cytometry. After incubation with serial antibodydilutions, staining of the cells is quantified by flow cytometricanalysis.

13. Epitope Binning

Epitope binning experiments were done using flow cytometry. Kit 225cells were incubated with a 100-fold excess of Fab-fragments and EC50concentrations of IgGs. The IgGs binding to the cells were quantifiedwith PE-labelled Fc-specific secondary antibody by Flowcytometry.Competition between the Fab and IgG for the same epitope or binding tothe same displayed surface in a close-by area (same bin) resulted indiminished detection of the IgG.

14. Size Exclusion Chromatography Coupled with Multi-Angle LightScattering Detector (SEC-MALS)

SEC-MALS measurements were performed on an Agilent 1200 HPLC system(Agilent Technologies) connected to a tri-angle light scatteringdetector (miniDAWN Treos, Wyatt Technology, Santa Barbara, Calif., USA).The concentration of the sample was followed online with a differentialrefractometer (Optilab rEX, Wyatt Technology) using a specificrefractive index increment (dn/dc) value of 0.186 ml/g (Wen et al.,1996). Sample volumes of 50 μl were injected on a Superdex 200 10/300 GLcolumn (GE Healthcare). The data were recorded and processed using theASTRA V software (Wyatt Technology). To determine the detector delayvolumes and normalization coefficients for the MALS detector, a BSAsample (Sigma, A8531) was used as reference. Neither despiking nor aband broadening correction was applied.

See also Wen, J., Arakawa, T., Philo, J. S., 1996. Anal. Biochem. 240,155-166.

15. In Process Control of Antibody Concentrations in Cell Supernatants(Titer)

Concentration determinations of antibody-containing supernatants wereperformed on a HP1100 HPLC system (Agilent Technologies) employingprotein A column. 62.5p1 protein A sepharose 4 fast flow (AmershamBiosciences) was packed into a HPLC cartridge. Running buffers were 50mM H₃BO₃, 200 mM Na₂SO₄, where buffer A was adjusted to pH7.5 using NaOHand buffer B to pH2.5 using H₂SO₄. The flow rate was 0.75 ml/min.Typically, 0.2 ml of supernatant was injected onto the protein A column.After rinsing with buffer A for 5.5 min, the bound antibody was elutedwith buffer B for 2.5 min. The column was equilibrated with buffer Aprior to the next sample injection. The 215 nm UV trace was monitoredand the elution peak area or height was used for quantitation. Astandard curve obtained from samples with different amounts of purifiedantibodies spiked into culture medium was used for calibration.

16. In Silico Prediction of the Isoelectric Point of Human Antibodies(p1)

The in-silico predicted μl was calculated by analyzing the primary aminoacid sequence of the full antibody with the Novartis Biologics μlCalculator version 1.1 developed at NBx-PSP-CPD by Markus Heitzmann,Tina Buch, Salvatore Leonardi, Nora Eifler and Michael Vetsch. pK valuesare from Grimsley G R., Scholtz J M, and Pace C N. (2009) Protein Sci.2009 18:247-51.

17. Protein A Recovery (%)

A multiwavelength nanodrop spectrophotometer ND1000 (Peglab) was used todetermine the concentration of the sample antibody after Protein Apurification at a wavelength of 280 nm. Protein A recovery is the ratioof the total amount of purified antibody and amount of antibody in thecell supernatant measured by Protein A HPLC (see above).

18. Melting Point Determination of IgG's at pH7.4

The system used was a Thermofluor® iQ™ 5 Optical System (BioRadLaboratories) and the software used was the iQ™ 5 Optical System v2.0.The 96-well white microplates (Multiplate® PCR Plate™) were from Biorad(#MPL9561). The plates were sealed with a Microseal® ‘B’ Film. TheSYPRO® Orange Protein Gel Stain was from Sigma (#S5692). Theconcentrated dye was diluted in dH2O prior to use (1.4 μl in 1 ml dH2O).10 μl of antibody sample were added to 10 μl of 250 mM Sodiumphosphatebuffer pH 7.4, 23 μl dH2O and 7 μl of the diluted dye. The final volumewas 50 μl per well. The plates were sealed with a Microseal® ‘B’ Filmusing a handheld sealer (avoiding fingerprints) and the plates werecentrifuged for 2 min at 1000g. Tm measurements were done in theThermofluor instrument by increasing the temperature from 20° C. until95° C. in 0.5° C. increments during 90 min.

19. Melting Point Determination of Fabs at pH7.4, pH6.0, pH 3.5

The system used was a Thermofluor® iQ™ 5 Optical System (BioRadLaboratories) and the software used was the iQ™ 5 Optical System v2.0.The 96-well white microplates (Multiplate® PCR Plate™) were from Biorad(#MPL9561). The plates were sealed with a Microseal® ‘B’ Film. TheSYPRO® Orange Protein Gel Stain was from Sigma (#S5692). Theconcentrated dye was diluted in dH2O prior to use (1.4 μl in 1 ml dH2O).25p1 of E. coli expressed Fab sample were added to 18p1 of 50 mM citratepH 3.5 or 100 mM MES pH6.0 or PBS and finally 7p1 of the diluted dyewere included. The final volume was 50 μl per well. Plates were sealedwith Microseal ‘B’ Adhesive Seals (BioRad, #MSB1001) using sealing tool,was mixed and spun down. Melting temperatures were measured by heatingsamples in an iQ™ 5 Multicolor Real Time PCR Detection System from 20 to100° C. in increments of 0.5° C. Results were analyzed using iQ5.

20. Amide Hydrogen/Deuterium Exchange Mass Spectrometry Mapping ofEpitopes

Amide Hydrogen/Deuterium exchange Mass Spectrometry (H/DxMS) was used toprobe IL12Rbeta1 for information regarding the epitope for the followinggroups of antibodies: (i) mAb1, mAb2, mAb3, mAb13 (MOR11873), (ii) mAb4,mAb5, mAb6, mAb14 (MOR11878), and (iii) mAb10, mAb11, mAb12, mAb16(MOR11880). H/DxMS mapping relies upon the mass difference between theisotopic masses of ¹H and ²H (also called deuterium and abbreviated withthe atom symbol D) or heavy hydrogen. Upon transfer from water (H₂O) toa heavy water (D₂O), a protein will experience an increase in mass asthe protein's hydrogen atoms become gradually replaced with deuterons(i.e. the heavier isotope of hydrogen). The likelihood of ahydrogen/deuterium exchange events is largely determined by proteinstructure under physiological conditions. The structure controls thethermodynamic stability of the protein at a given site and thesite-specific solvent accessibility. H/DxMS technology is used tomeasure the mass shifts of proteolytic fragments of the protein thatresult from deuterium incorporation into the protein under physiologicalconditions. These mass shifts indirectly report back on the localprotein structural features (geometric and energetic). Of theexchangeable hydrogens in a protein only the amide hydrogens areobserved in an H/DxMS analysis as it is not possible to stabilize thedeuteration at other sites sufficiently to survive post-labelinganalysis.

When a binding partner binds to an antibody target (e.g.antigen/antibody interaction), experimentally changes inhydrogen/deuterium exchange rate may be observed as a result of solventexclusion and energetic stabilization of the local environment. Surfaceregions that are excluded from solvent access upon complex formationexchange much more slowly. Reduced exchange can therefore be directlyused as an indication for a binding site. Specifically, inantigen-antibody interactions these changes might highlight the locationof the epitope. This interpretation is complicated by the fact thatchanges (geometric and energetic) can occur elsewhere in the protein(allosteric changes) as the protein responds in cooperative manner toligand binding. Allosteric effects on hydrogen-deuterium exchange cannotbe discriminated against in a mass spectrometry based assay as nogeometric/structural information is collected. Nevertheless, it isobserved in practice that solvent exclusion explains mostly theprotection patterns observed in protein-antigen complex formation asreferenced toward the free antigen and that locations on the proteinshowing reduced exchange are highly indicative of a binding interface.

The location of reduced deuteron incorporation after antibody bindingmay be deduced by digestion of the target protein followinghydrogen/deuterium exchange (e.g. with a suitable enzyme such as pepsin)and then mass spectrometry to determine the mass shift of the relevantfragments.

Experimental description: In the labeling experiment, the antigen(IL12Rbeta1) and the antigen-antibody complex were on-exchanged insolution at 0° C. for a predetermined time using an automated liquidhandling system.

After the in-exchange period of 10 min samples are quenched to low pH(approx. 2.5 as this slows the exchange process of amide-hydrogens byfive orders of magnitude as compared to pH 7.5) to stop the exchange andpreserve the labeled state by addition of an excess of acidic quenchbuffer. Quenching to low pH provides enough stabilization of the labeledstate (half-life ˜30 min) for proteolysis (pepsin) and analysis byliquid chromatography mass spectrometry.

The differences in deuteration for the fragments from theantibody-antigen complex and the free antigen are inspected. Negativevalues are indicative of solvent protection in the antibody-antigencomplex and interpreted as the most likely sites of interaction.

The results of this analysis, when conducted with representativeantibodies from three groups of antibodies having the MOR# variableregions MOR11873, MOR11878, MOR11880 as set out in table 13, areprovided in table 15 below.

Examples mAb1 to mAb16

Table 6 describes the amino acid sequences (SEQ ID NOs) of the fulllength heavy and light chains of examples mAb1 to mAb16.

TABLE 6 Examples mAb1-mAb16 Full Length Heavy Chain Full Length LightChain Antibody Amino acid sequence Amino acid sequence mAb1 SEQ ID NO:57 SEQ ID NO: 69 mAb2 SEQ ID NO: 61 SEQ ID NO: 69 mAb3 SEQ ID NO: 65 SEQID NO: 69 mAb4 SEQ ID NO: 58 SEQ ID NO: 70 mAb5 SEQ ID NO: 62 SEQ ID NO:70 mAb6 SEQ ID NO: 66 SEQ ID NO: 70 mAb7 SEQ ID NO: 59 SEQ ID NO: 71mAb8 SEQ ID NO: 63 SEQ ID NO: 71 mAb9 SEQ ID NO: 67 SEQ ID NO: 71 mAb10SEQ ID NO: 60 SEQ ID NO: 72 mAb11 SEQ ID NO: 64 SEQ ID NO: 72 mAb12 SEQID NO: 68 SEQ ID NO: 72 mAb13 SEQ ID NO: 90 SEQ ID NO: 69 mAb14 SEQ IDNO: 91 SEQ ID NO: 70 mAb15 SEQ ID NO: 92 SEQ ID NO: 71 mAb16 SEQ ID NO:93 SEQ ID NO: 72

The Examples mAb1 to mAb16 can be produced using conventional antibodyrecombinant production and purification processes. For example, thecoding sequences as defined in Table 7 are cloned into a productionvector for recombinant expression in mammalian production cell line.

TABLE 7 Coding DNA sequences of mAb1-mAb16 Full Length Heavy Chain FullLength Light Chain Example DNA coding sequence DNA coding sequence mAb1SEQ ID NO: 73 SEQ ID NO: 85 mAb2 SEQ ID NO: 77 SEQ ID NO: 85 mAb3 SEQ IDNO: 81 SEQ ID NO: 85 mAb4 SEQ ID NO: 74 SEQ ID NO: 86 mAb5 SEQ ID NO: 78SEQ ID NO: 86 mAb6 SEQ ID NO: 82 SEQ ID NO: 86 mAb7 SEQ ID NO: 75 SEQ IDNO: 87 mAb8 SEQ ID NO: 79 SEQ ID NO: 87 mAb9 SEQ ID NO: 83 SEQ ID NO: 87mAb10 SEQ ID NO: 76 SEQ ID NO: 88 mAb11 SEQ ID NO: 80 SEQ ID NO: 88mAb12 SEQ ID NO: 84 SEQ ID NO: 88 mAb13 SEQ ID NO: 94 SEQ ID NO: 85mAb14 SEQ ID NO: 95 SEQ ID NO: 86 mAb15 SEQ ID NO: 96 SEQ ID NO: 87mAb16 SEQ ID NO: 97 SEQ ID NO: 88

Other antibodies retaining substantially the same binding properties toIL12Rβ1 includes chimeric antibodies of any one of mAb1 to mAb16 whichretain the same V_(H) and V_(L) regions of any one of mAb1 to mAb16 anddifferent constant regions (for example a different Fc region selectedfrom a different isotype, for example IgG4 or IgG2).

Table 8 summarizes the variable heavy (V_(H)) and light chain (V_(L))amino acid sequence of mAb1 to mAb16 which can be used to generatechimeric antibodies from mAb1 to mAb16.

TABLE 8 Variable heavy (V_(H)) and light chain (V_(L)) amino acidsequence Variable Heavy Chain Variable Light Chain Original antibodyAmino acid sequence Amino acid sequence mAb1, mAb2, mAb3, SEQ ID NO: 49SEQ ID NO: 50 mAb13 mAb4, mAb5, mAb6, SEQ ID NO: 51 SEQ ID NO: 52 mAb14mAb7, mAb8, mAb9, SEQ ID NO: 53 SEQ ID NO: 54 mAb15 mAb10, mAb11, SEQ IDNO: 55 SEQ ID NO: 56 mAb12, mAb16

Other example antibodies, retaining substantially the same bindingproperties to IL12Rβ1 as mAb1-mAb16, include CDR grafted antibodies ofany one of mAb1 to mAb16 by CDR grafting, retaining the CDR regions ofany one of mAb1 to mAb16 with different framework and/or constantregions.

Table 9 summarizes the useful CDR sequences of mAb1 to mAb16 to generatealternative CDR grafted antibodies, wherein the CDR regions from mAb1 tomAb16 are defined according to Kabat definition.

Table 10 summarizes the useful CDR sequences of mAb1 to mAb16 togenerate alternative CDR grafted antibodies, wherein the CDR regionsfrom mAb1 to mAb16 are defined according to Chothia definition.

TABLE 9 CDR regions from mAb1 to mAb16 are defined according to Kabatdefinition Original antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 mAb1,SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb2, NO: 1 NO: 2 NO: 3 NO: 4NO: 5 NO: 6 mAb3, mAb13 mAb4, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDmAb5, NO: 7 NO: 8 NO: 9 NO: 10 NO: 11 NO: 12 mAb6, mAb14 mAb7, SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb8, NO: 13 NO: 14 NO: 15 NO: 16 NO:17 NO: 18 mAb9, mAb15 mAb10, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDmAb11, NO: 19 NO: 20 NO: 21 NO: 22 NO: 23 NO: 24 mAb12, mAb16

TABLE 10 CDR regions from mAb1 to mAb16 according to Chothia definitionOriginal anti- body HCDR1′ HCDR2′ HCDR3′ LCDR1′ LCDR2′ LCDR3′ mAb1, SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb2, NO: 25 NO: 26 NO: 27 NO: 28NO: 29 NO: 30 mAb3, mAb13 mAb4, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID mAb5, NO: 31 NO: 32 NO: 33 NO: 34 NO: 35 NO: 36 mAb6, mAb14 mAb7, SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID mAb8, NO: 37 NO: 38 NO: 39 NO: 40NO: 41 NO: 42 mAb9, mAb15 mAb10, SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID mAb11, NO: 43 NO: 44 NO: 45 NO: 46 NO: 47 NO: 48 mAb12, mAb16Generation of Anti-IL12Rbeta1 mAbs

Once a single, archetypal anti-human-IL12Rbeta1 antibody, for exampleany one of mAbs 1 to 16, has been isolated that has the desiredproperties described herein it is straightforward to generate otherantibodies with similar properties, by using art-known methods. Forexample, mice may be immunized with IL12Rbeta1, hybridomas produced, andthe resulting antibodies screened for binding to the IL12Rbeta1polypeptide of SEQ ID NO:89. Alternatively, phage display methodologiesmay be used to generate other antibodies having specificity for theIL12Rbeta1 polypeptide of SEQ ID NO:89.

Such other antibodies having specificity for the IL12Rbeta1 polypeptideof SEQ ID NO:89 can then be screened for one or more of the followingproperties: a KD of 1 nM or less using Surface Plasmon Resonance(Biacore system), inhibition of IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay, the ability to compete with the archetypalmAb for binding to IL12Rbeta1, as measured using Surface PlasmonResonance (Biacore system) or Elisa-based cross-blocking assay, bindingwithin or to the same epitope, as determined using amideHydrogen/Deuterium exchange Mass Spectrometry. Alternatively, the methodof Jespers et al., Biotechnology 12:899, 1994, which is incorporatedherein by reference, may be used to guide the selection of mAbs havingthe same epitope and therefore similar properties to the archetypal mAb,e.g., one of any one of mAbs 1 to 16. Using phage display, first theheavy chain of the archetypal antibody is paired with a repertoire of(preferably human) light chains to select an IL12Rbeta1-binding mAb, andthen the new light chain is paired with a repertoire of (preferablyhuman) heavy chains to select a (preferably human) IL12Rbeta1-bindingmAb having the same epitope as the archetypal mAb.

For example, to identify other antibodies binding within or to theepitope described herein, the antibodies can be generated as describedabove, and then screened for binding to or within the epitope usingamide Hydrogen/Deuterium exchange Mass Spectrometry. Alternatively, toidentify those other antibodies which compete with the archetypal mAbfor binding to IL12Rbeta1, the antibodies can be generated as describedabove, and then screened for competition using Surface Plasmon Resonance(Biacore system) or Elisa-based cross-blocking assay.

To identify other antibodies having the functional properties describedherein, the antibodies can be generated as described above, and thenscreened for a KD of 1 nM or less using Surface Plasmon Resonance(Biacore system), and inhibition of IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay.

To identify other antibodies having the functional properties describedherein and which bind within or to the epitope described herein, theantibodies can be generated as described above, screened for a KD of 1nM or less using Surface Plasmon Resonance (Biacore system), andinhibition of IL12 and/or IL23 binding to the IL12Rbeta1 polypeptide ofSEQ ID NO:89 as measured in an in vitro competitive binding assay, andscreened for binding to or within the epitope using amideHydrogen/Deuterium exchange Mass Spectrometry.

To identify other antibodies having the functional properties describedherein and which compete with the archetypal mAb for binding toIL12Rbeta1, the antibodies can be generated as described above, screenedfor a KD of 1 nM or less using Surface Plasmon Resonance (Biacoresystem), screened for inhibition of IL12 and/or IL23 binding to theIL12Rbeta1 polypeptide of SEQ ID NO:89 as measured in an in vitrocompetitive binding assay, and screened for competition using SurfacePlasmon Resonance (Biacore system) or Elisa-based cross-blocking assay.

TABLE 11 Brief description of useful amino acid and nucleotide sequencesfor practicing the invention SEQ ID NO: Description of the sequence 1HCDR1 amino acid sequence of MOR11873 according to Kabat definition 2HCDR2 amino acid sequence of MOR11873 according to Kabat definition 3HCDR3 amino acid sequence of MOR11873 according to Kabat definition 4LCDR1 amino acid sequence of MOR11873 according to Kabat definition 5LCDR2 amino acid sequence of MOR11873 according to Kabat definition 6LCDR3 amino acid sequence of MOR11873 according to Kabat definition 7HCDR1 amino acid sequence of MOR11878 according to Kabat definition 8HCDR2 amino acid sequence of MOR11878 according to Kabat definition 9HCDR3 amino acid sequence of MOR11878 according to Kabat definition 10LCDR1 amino acid sequence of MOR11878 according to Kabat definition 11LCDR2 amino acid sequence of MOR11878 according to Kabat definition 12LCDR3 amino acid sequence of MOR11878 according to Kabat definition 13HCDR1 amino acid sequence of MOR11879 according to Kabat definition 14HCDR2 amino acid sequence of MOR11879 according to Kabat definition 15HCDR3 amino acid sequence of MOR11879 according to Kabat definition 16LCDR1 amino acid sequence of MOR11879 according to Kabat definition 17LCDR2 amino acid sequence of MOR11879 according to Kabat definition 18LCDR3 amino acid sequence of MOR11879 according to Kabat definition 19HCDR1 amino acid sequence of MOR11880 according to Kabat definition 20HCDR2 amino acid sequence of MOR11880 according to Kabat definition 21HCDR3 amino acid sequence of MOR11880 according to Kabat definition 22LCDR1 amino acid sequence of MOR11880 according to Kabat definition 23LCDR2 amino acid sequence of MOR11880 according to Kabat definition 24LCDR3 amino acid sequence of MOR11880 according to Kabat definition 25HCDR1′ amino acid sequence of MOR11873 according to Chothia definition26 HCDR2′ amino acid sequence of MOR11873 according to Chothiadefinition 27 HCDR3′ amino acid sequence of MOR11873 according toChothia definition 28 LCDR1′ amino acid sequence of MOR11873 accordingto Chothia definition 29 LCDR2′ amino acid sequence of MOR11873according to Chothia definition 30 LCDR3′ amino acid sequence ofMOR11873 according to Chothia definition 31 HCDR1′ amino acid sequenceof MOR11878 according to Chothia definition 32 HCDR2′ amino acidsequence of MOR11878 according to Chothia definition 33 HCDR3′ aminoacid sequence of MOR11878 according to Chothia definition 34 LCDR1′amino acid sequence of MOR11878 according to Chothia definition 35LCDR2′ amino acid sequence of MOR11878 according to Chothia definition36 LCDR3′ amino acid sequence of MOR11878 according to Chothiadefinition 37 HCDR1′ amino acid sequence of MOR11879 according toChothia definition 38 HCDR2′ amino acid sequence of MOR11879 accordingto Chothia definition 39 HCDR3′ amino acid sequence of MOR11879according to Chothia definition 40 LCDR1′ amino acid sequence ofMOR11879 according to Chothia definition 41 LCDR2′ amino acid sequenceof MOR11879 according to Chothia definition 42 LCDR3′ amino acidsequence of MOR11879 according to Chothia definition 43 HCDR1′ aminoacid sequence of MOR11880 according to Chothia definition 44 HCDR2′amino acid sequence of MOR11880 according to Chothia definition 45HCDR3′ amino acid sequence of MOR11880 according to Chothia definition46 LCDR1′ amino acid sequence of MOR11880 according to Chothiadefinition 47 LCDR2′ amino acid sequence of MOR11880 according toChothia definition 48 LCDR3′ amino acid sequence of MOR11880 accordingto Chothia definition 49 V_(H) amino acid sequence of MOR11873 50 V_(L)amino acid sequence of MOR11873 51 V_(H) amino acid sequence of MOR1187852 V_(L) amino acid sequence of MOR11878 53 V_(H) amino acid sequence ofMOR11879 54 V_(L) amino acid sequence of MOR11879 55 V_(H) amino acidsequence of MOR11880 56 V_(L) amino acid sequence of MOR11880 57 Fulllength heavy chain of MOR11873 with IgG1 LALA Fc variant 58 Full lengthheavy chain of MOR11878 with IgG1 LALA Fc variant 59 Full length heavychain of MOR11879 with IgG1 LALA Fc variant 60 Full length heavy chainof MOR11880 with IgG1 LALA Fc variant 61 Full length heavy chain ofMOR11873 with IgG1 D265A Fc variant 62 Full length heavy chain ofMOR11878 with IgG1 D265A Fc variant 63 Full length heavy chain ofMOR11879 with IgG1 D265A Fc variant 64 Full length heavy chain ofMOR11880 with IgG1 D265A Fc variant 65 Full length heavy chain ofMOR11873 with IgG1 N297A Fc variant 66 Full length heavy chain ofMOR11878 with IgG1 N297A Fc variant 67 Full length heavy chain ofMOR11879 with IgG1 N297A Fc variant 68 Full length heavy chain ofMOR11880 with IgG1 N297A Fc variant 69 Full length light chain ofMOR11873 70 Full length light chain of MOR11878 71 Full length lightchain of MOR11879 72 Full length light chain of MOR11880 73 Nucleotidesequence encoding Full length heavy chain of MOR11873 with IgG1 LALA Fcvariant 74 Nucleotide sequence encoding Full length heavy chain ofMOR11878 with IgG1 LALA Fc variant 75 Nucleotide sequence encoding Fulllength heavy chain of MOR11879 with IgG1 LALA Fc variant 76 Nucleotidesequence encoding Full length heavy chain of MOR11880 with IgG1 LALA Fcvariant 77 Nucleotide sequence encoding Full length heavy chain ofMOR11873 with IgG1 D265A Fc variant 78 Nucleotide sequence encoding Fulllength heavy chain of MOR11878 with IgG1 D265A Fc variant 79 Nucleotidesequence encoding Full length heavy chain of MOR11879 with IgG1 D265A Fcvariant 80 Nucleotide sequence encoding Full length heavy chain ofMOR11880 with IgG1 D265A Fc variant 81 Nucleotide sequence encoding Fulllength heavy chain of MOR11873 with IgG1 N297A Fc variant 82 Nucleotidesequence encoding Full length heavy chain of MOR11878 with IgG1 N297A Fcvariant 83 Nucleotide sequence encoding Full length heavy chain ofMOR11879 with IgG1 N297A Fc variant 84 Nucleotide sequence encoding Fulllength heavy chain of MOR11880 with IgG1 N297A Fc variant 85 Nucleotidesequence encoding Full length light chain of MOR11873 86 Nucleotidesequence encoding Full length light chain of MOR11878 87 Nucleotidesequence encoding Full length light chain of MOR11879 88 Nucleotidesequence encoding Full length light chain of MOR11880 89 Amino acidsequence of human IL12Rbeta1 90 Full length heavy chain of MOR11873 withIgG1 Fc wild type 91 Full length heavy chain of MOR11878 with IgG1 Fcwild type 92 Full length heavy chain of MOR11879 with IgG1 Fc wild type93 Full length heavy chain of MOR11880 with IgG1 Fc wild type 94Nucleotide sequence encoding Full length heavy chain of MOR11873 withwild type IgG1 95 Nucleotide sequence encoding Full length heavy chainof MOR11878 with wild type IgG1 96 Nucleotide sequence encoding Fulllength heavy chain of MOR11879 with wild type IgG1 97 Nucleotidesequence encoding Full length heavy chain of MOR11880 with wild typeIgG1 98 Cynomolgous IL12Rβ1 amino acid sequence 99 Human p40 subunitamino acid sequence 100 Human p35 subunit amino acid sequence 101 Humanp19 subunit amino acid sequence

TABLE 12 Useful amino acid and nucleotide sequences for practicing the inventionSEQ ID NO: Describes the amino acid or nucleotide sequence below: 1SYGMS 2 GISYSGSDTEYADSVKG 3 SPDYIIDYGFDY 4 RASQGISSDLA 5 DASSLQS 6QQYWIYPFT 7 SYGMS 8 GISYDASDTEYADSVKG 9 SPDYIIDYGFDY 10 RASQGISSDLA 11DASSLQS 12 QQYWWYPFT 13 GYYMH 14 MIGPQHGEAIYAQKFQG 15 ESTDSDESPFDY 16SGDNIRSYYVS 17 DDSDRPS 18 QSYGSHSNFVV 19 GYYMH 20 MIGPQHGEAIYAQKFQG 21ESTDSDESPFDY 22 SGDNIRSYYVS 23 DDSDRPS 24 QSYGSHSNFVV 25 GFTFTSY 26SYSGSD 27 SPDYIIDYGFDY 28 SQGISSD 29 DAS 30 YWIYPF 31 GFTFTSY 32 SYDASD33 SPDYIIDYGFDY 34 SQGISSD 35 DAS 36 YWWYPF 37 GYTFTGY 38 GPQHGE 39ESTDSDESPFDY 40 DNIRSYY 41 DDS 42 YGSHSNFV 43 GYTFTGY 44 GPQHGE 45ESTDSDESPFDY 46 DNIRSYY 47 DDS 48 YGSHSNFV 49QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYSGSDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSS 50AIQMTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWIYPFTFGQGTK VEIK 51QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYDASDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSS 52AIQMTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWWYPFTFGQGT KVEIK 53QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARESTDSDESPFDYWGQGTLVTVSS 54DIELTQPPSVSVSPGQTASITCSGDNIRSYYVSWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYGSHSNFVVFGGGT KLTVL 55QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARESTDSDESPFDYWGQGTLVTVSS 56SYELTQPLSVSVALGQTARITCSGDNIRSYYVSWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQSYGSHSNFVVFGGGT KLTVL 57QVQLVESGGGWQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYSGSDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 58QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYDASDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 59QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 60QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 61QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYSGSDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 62QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYDASDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 63QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 65QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYSGSDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 66QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYDASDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 67QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 68QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 69AIQMTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWIYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 70AIQMTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWWYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 71DIELTQPPSVSVSPGQTASITCSGDNIRSYYVSWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYGSHSNFVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS 72SYELTQPLSVSVALGQTARITCSGDNIRSYYVSWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQSYGSHSNFVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS 73caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgccgccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacagcggcagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagcagcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 74caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacgacgccagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagcagcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcat gatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaaca gcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc gagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggac aagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca cgcagaagagcctctccctgtctccgggtaaa 75caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggctagtgtgaaggtgtcctgcaagg ccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgaggacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactggggccagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagcagcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 76caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggccagcgtgaaggtgtcctgcaag gccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgacgacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactgggggcagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagcagcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagt gcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccc cgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgac ctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaa caactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgga caagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactac acgcagaagagcctctccctgtctccgggtaaa 77caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacagcggcagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggccgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 78caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacgacgccagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagc ccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacatgcgtggtggtggccgtgagccacgaagaccctgaggtcaagttca actggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag cacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccg agaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaaca actacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacac gcagaagagcctctccctgtctccgggtaaa 79caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggctagtgtgaaggtgtcctgcaagg ccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgaggacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactggggccagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggccgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 80caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggccagcgtgaaggtgtcctgcaag gccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgacgacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactgggggcagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaag cccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccacc gtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctca tgatctcccggacccctgaggtcacatgcgtggtggtggccgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaaca gcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc gagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggac aagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca cgcagaagagcctctccctgtctccgggtaaa 81caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacagcggcagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 82caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgccgccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacgacgccagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagc ccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttca actggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccag cacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccg agaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaaca actacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacac gcagaagagcctctccctgtctccgggtaaa 83caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggctagtgtgaaggtgtcctgcaagg ccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgaggacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactggggccagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 84caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggccagcgtgaaggtgtcctgcaag gccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgacgacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactgggggcagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 85gccatccagatgacccagagccccagcagcctgagcgccagcgtgggcgacagagtgaccatcacctg tcgggccagccagggcatcagcagcgacctggcctggtatcagcagaagcccggcaaggcccccaag ctgctgatctacgacgccagctccctgcagagcggcgtgcccagcagattttccggcagcggctccggca ccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtactgg atctaccccttcaccttcggccagggcaccaaggtggaaatcaagcgtacggtggccgctcccagcgtgtt catcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaacttc tacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggaa agcgtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctgagcaagg ccgactacgagaagcacaaggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgacc aagagcttcaaccggggcgagtgt 86gccatccagatgacccagagccccagcagcctgagcgccagcgtgggcgacagagtgaccatcacctg tcgggccagccagggcatcagcagcgacctggcctggtatcagcagaagcccggcaaggcccccaagctgctgatctacgacgccagctccctgcagagcggcgtgcccagcagattttccggcagcggctccggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacttactactgccagcagtactggtggtatcccttcaccttcggccagggcaccaaggtggaaatcaagcgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggaaagcgtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaaccggggcgagtgt 87gacatcgagctgacccagccccctagcgtgtccgtgtctcctggccagaccgccagcatcacctgtagcggcgacaacatcagatcctactacgtgtcctggtatcagcagaagcccggccaggcccccgtgctggtcatctacgacgacagcgaccggcccagcggcatccccgagagattcagcggcagcaacagcggcaacaccgccaccctgaccatcagcggcacccaggccgaggacgaggccgactactactgccagagctacggcagccacagcaacttcgtggtgttcggcggaggcaccaagttaaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 88tcttacgagctgactcagcccctgtccgtgtctgtggctctgggccagaccgcccggatcacatgcagcggc gacaacatcagatcctactacgtgtcctggtatcagcagaagcctggacaggcccccgtgctggtcatctac gacgacagcgaccggcccagcggcatccccgagagattcagcggaagcaacagcggcaacaccgcc accctgaccatctccagagcccaggccggcgacgaggccgactactactgccagagctacggcagcca cagcaacttcgtggtgttcggcggaggcaccaagttaaccgtcctaggtcagcccaaggctgccccctcgg tcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttc tacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagacca ccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcag tggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 89 MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSDWLIFFASLGSFLSILLVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM 90QVQLVESGGGWQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYSGSDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  91QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVAGISYDASDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPDYIIDYGFDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  92QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  93QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPQHGEAIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARESTDSDESPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 94caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacagcggcagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 95caggtgcaattggtggaaagcggcggaggcgtggtgcagcctggcagaagcctgagactgagctgtgcc gccagcggcttcaccttcaccagctacggcatgagctgggtccgacaggcccctggcaagggcctggaatgggtggccggcatcagctacgacgccagcgacaccgagtacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaagccccgactacatcatcgactacggcttcgactactggggcagaggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagc ccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttca actggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag cacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccg agaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaaca actacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacac gcagaagagcctctccctgtctccgggtaaa 96caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggctagtgtgaaggtgtcctgcaagg ccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgaggacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactggggccagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 97caggtgcaattggtgcagtctggcgccgaagtgaagaaacctggggccagcgtgaaggtgtcctgcaag gccagcggctacaccttcaccggctactacatgcactgggtccgacaggcccctggacagggcctggaatggatgggcatgatcggcccccagcacggcgaggccatctacgcccagaaattccagggcagagtgaccatgacccgggacaccagcatcagcaccgcctacatggaactgagccggctgcggagcgacgacaccgccgtgtactactgcgccagagagagcaccgacagcgacgagagccccttcgactactgggggcagggcaccctggtcaccgtcagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaag cccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccacc gtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctca tgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaaca gcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc gagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggac aagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca cgcagaagagcctctccctgtctccgggtaaa 98 MEPLVTWVVPLLLLFLRSRQGAACGTSECCFQDPPYSDADSGSASGPRDLSCYRISSAGYECSWQYEGPTAGVSHFLRCCLSSGRCCYFATGSATRLQFSDQAGVSVLHTVTLWVESWARNRTEKSPEVTLQLYKSVKYKPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSSWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGRDGRRRLTLKEQPTQLELPEGCQGPAPGVEVTYQLQLHMLSCPCKAKATRTLPLEKMPYLSGAAYNVAVISSNRFGPGPNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSTTYCIEWQPVGQEGSLATCNLTAPQDPDPAGMATYSWSRESGAMGQEKCYHITIFASAHPKKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSNQVSEHPVQPTETQVTLSDLRAGVAYTVQVRADTA WLRGAWSQPQRFSIKVQVSDWFIFFASLGSFLSILLVGVLGYLGLNRATRHLCPPLPTPCASSAIQFPAGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTELLEKAELPEGAPE LALDTQLSLEDGDRCKAKM99 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS 100MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLD HLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS 101 MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDV PHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVA VAARVFAHGAATLSPResults

Examples mAb1 to mAb16 were obtained and produced as described below:

Screening and Identification of Fab Antibodies with High Affinity toIL12Rb1

Library screening combined with rapid maturation was performed asdescribed (Knappik et al., 2000 J. Mol. Biol. 296: 57-86; Pressler etal., 2009 Immunotherapy 1(4): 571-83). In addition to the classicalscreening confirmed binders (Fab) were immediately tested for binding onhuman (Kit-225) and cyno (HSC-F) cells naturally expressing the IL-12R.This allowed the selection of 119 unique clones from the originallyobserved 5520 hits. They were then tested for functional inhibition inthe human whole blood assay, followed by the cyno version for effectivecandidates (46 from 20 families, falling into 4 epitope bins). Thus 6efficacious functional inhibitors (from 4 families) were initiallyshortlisted due to their IC₅₀ inhibiting cyno and human IL-12 functions.The top 4 (designated MOR11873, MOR11878, MOR11879 and MOR11880respectively; the two latter being two different frameworks containingidentical CDRs) were selected for optimization engineering to improvetheir developability and prepared in four different IgG1 Fc versions forthe assessment of the optimal silent version (ADCC/CDC pending).

Expression and Purification of HuCAL®-Fab Antibodies in E. coli

To facilitate rapid expression of soluble Fab, the Fab encoding insertsof the selected HuCAL PLATINUM® phage were subcloned into an expressionvector for IPTG inducible Fab expression in mammalian cell lines andpurification of the Fab by Ni-NTA chromatography. Expression of Fabfragments in TG-1 cells was induced by addition of IPTG. Cells weredisrupted using lysozyme and Fab fragments isolated by Ni-NTAchromatography (Bio-Rad, Germany). Protein concentrations weredetermined by UV-spectrophotometry. Purity of Fab fragments was analyzedin denatured, reduced state using SDS-PAGE and in native state byHP-SEC.

Conversion to IgG

In order to express full length IgG, variable domain fragments of heavy(V_(H)) and light chains (V_(L)) of the four lead candidates MOR11873,MOR11878, MOR11879 and MOR11880 were subcloned from Fab expressionvectors into appropriate expression vectors for human IgG1f wild type,human IgG1fD265A, human IgG1fN297A, and human IgG1f L234AL235A hereafterreferred as “LALA”, resulting in expression vectors for the productionof the 16 antibodies according to the invention, mAb1-mAb16 as describedin Table 13 below:

TABLE 13 Description of the variable regions and IgG1 Fc region ofmAb1-mAb16 Example MOR# variable region and IgG1 Fc region mAb1 MOR11873with IgG1 LALA Fc variant mAb2 MOR11873 with IgG1 D265A Fc variant mAb3MOR11873 with IgG1 N297A Fc variant mAb4 MOR11878 with IgG1 LALA Fcvariant mAb5 MOR11878 with IgG1 D265A Fc variant mAb6 MOR11878 with IgG1N297A Fc variant mAb7 MOR11879 with IgG1 LALA Fc variant mAb8 MOR11879with IgG1 D265A Fc variant mAb9 MOR11879 with IgG1 N297A Fc variantmAb10 MOR11880 with IgG1 LALA Fc variant mAb11 MOR11880 with IgG1 D265AFc variant mAb12 MOR11880 with IgG1 N297A Fc variant mAb13 MOR11873 withIgG1 wild type Fc mAb14 MOR11878 with IgG1 wild type Fc mAb15 MOR11879with IgG1 wild type Fc mAb16 MOR11880 with IgG1 wild type FcTransient Expression and Purification of Human IgG

Eukaryotic HKB11 cells were transfected with expression vector DNAencoding for heavy and light chains of IgGs mAb1, mAb4, mAb7 and mAb10.After sterile filtration, the solution was subjected to standard proteinA affinity chromatography (MabSelect SURE, GE Healthcare). Proteinconcentrations were determined by UV-spectrophotometry. Purity of IgGwas analyzed under denaturing, reducing and non-reducing conditions inSDS-PAGE or by using Caliper LabChip® System or Agilent BioAnalyzer andin native state by HP-SEC. All candidate antibodies were tested fortheir IL12Rβ1 binding affinity (SPR, K_(D) nM), IL12/IL23 in vitrocompetitive binding inhibition, IL12/IL18 dependent IFNγ production inhuman and in cynomolgous monkeys according to the methods described inthe Method paragraph above. The results are shown in Table 14.

TABLE 14 Profiling data of mAb1, mAb4, mAb7 and mAb10 Selection CriteriamAb1 mAb4 mAb7 mAb10 human IL12Rβ1 binding affinity 40 20 40 40 (SPR,K_(D) pM) cyno IL12Rβ1 binding affinity 100 100 600 600 (SPR, K_(D) pM)Cell binding Kit225 6 5 10 11 (human natural expression; EC₅₀, pM) Cellbinding human T-cells 33 38 15 16 (human natural expression; EC₅₀, pM)Cell binding HSC-F 23 13 23 n.d. (cyno natural expression; EC₅₀, pM)Cell binding cyno T-cells 55 41 36 28 (cyno natural expression; EC₅₀,pM) IL12/IL23 competitive binding 8/18 8/13 8/20 8/20 (IC₅₀, pM)IL12/IL18 dependent IFNγ 28 77 24 42 production In human blood cells(IC₅₀, pM) IL12/IL18 dependent IFNγ 239 216 284 1182 production In cynoblood cells (IC₅₀, pM) Epitope bin (A-D) B B B B analytical SEC (%monomer) 97.8 98.5 98.0 98.7 titer (mg/L) 51.0 33.8 51.7 42.9 pI(in-silico prediction) 8.6 8.2 6.8 7.5 Tm (° C., pH 7.4, IgG data) 76.075.3 68.5 68.0 protein A recovery (%) 62.5 76.2 36.8 69.5 Tm (° C., pH7.4, Fab data) 68.0 54.5 66.5 66.3 Tm (° C., pH 6.0, Fab data) 73.0 74.569.0 69.5 Tm (° C., pH 3.5, Fab data) 56.8 56.0 55.8 55.8

Remarkably, the antibodies of the invention have K_(D) affinity and IC₅₀below 100 pM, and even below 10 pM for IL12 competitive binding asdetermined by the functional human assays, therefore being particularlysuitable for use as a drug. Moreover, they possess advantageousdevelopability properties. They also cross-react with cynomolgous monkeyIL12Rβ1 (SEQ ID NO:98) and the coding sequences encoding the variableregions can be easily transferred for generating silent IgG1 antibodies,for example comprising the IgG1 Fc variant containing the L234A L235Amutation, or the IgG1 Fc variant containing the D265A mutation, or theIgG1 Fc variant containing the N297A mutation.

Epitope Binning Experiment

The antibodies were tested in cross-competition (epitope binning)experiments and 4 distinct groups could be distinguished among alloriginally identified functionally inhibiting mAbs. The selected top 4candidates MOR11873, MOR11878, MOR11879 and MOR11880 all happen to fallinto the second group designated epitope bin B and thus compete witheach other. Since all mAb1-mAb16 have a binding region derived from oneof the four top candidates, it is therefore expected that all mAb1-mAb16antibodies compete with each other.

Summary of Amide Hydrogen/Deuterium Exchange Mass Spectrometry EpitopeMapping

In order to develop a dearer picture of the binding relationship betweenthe antibodies of the invention, epitope mapping studies were carriedout. The object was to determine the site of binding of the candidatemonoclonal antibodies on the target human IL12Rbeta1 molecule. inparticular, the objective was to identify the specific amino acidresidues of human IL12Rbeta1 that are involved in the bindinginteraction between the antibodies and target. Epitope mapping wasgenerally carried out using state of the art techniques available to theskilled person. In particular, Hydrogen/Deuterium exchange massspectrometry was used to probe the structure of human IL12Rbeta1 foramino acid residues which influence antibody binding, i.e. areimplicated in the relevant epitopes. The results in Table 15 show Δ(Delta) values, i.e. the difference in deuteration between theIL12Rbeta1/antibody complex and the free IL12Rbeta1 protein. Negativevalues are indicative of solvent protection in the antibody-antigencomplex and interpreted as the most likely sites of interaction.

TABLE 15  H/DxMS epitope mapping data Position of peptide in MOR11873MOR11878 MOR11880 IL12Rbeta 1 peptide sequence IL12Rbeta1 Δ (Delta) Δ(Delta) Δ (Delta) FAAGSATRLQFSDQAGVSVL  89-108 3.84 1.59 2.58TRLQFSDQAGVSVL  95-108 0.98 YNSVKYEPPLGDIKVSKLAGQL 134-155 1.91CSLTAPQDPDPAGM 383-396 0.36 TAPQDPDPAGMAT 386-398 0.05 0.04 0.03ITIFASAHPEKLTL 416-429 −0.37 −0.38 −0.48 LSTCPGVLKE 471-480 0.61 1.02YVVRCRDEDSKQ 481-492 −0.05 −0.04 SGLRAGVA 508-515 0.46 0.16 0.31YTVQVRADTAWLRGVWSQPQRF 516-537 0.81 VRADTAWLRGVWSQPQRF 520-537 0.99 0.240.15

The H/DxMS epitope mapping results show that one peptide correspondingto amino acid residues 416-429 of the human IL12Rbeta1 sequence(ITIFASAHPEKLTL) exhibited protection for all groups of antibodies. Thisdata is consistent with the fact that these groups of antibodies showcompetition with each other, indicating that the 416-ITIFASAHPEKLTL-429peptide corresponds to an epitope for all three groups of antibodies.Furthermore, these data are consistent with that fact that theantibodies are cross-reactive with cynomolgus IL12Rbeta1, but not theequivalent rodent sequences (mouse and rat). A comparison of thisepitope sequence in human, cynomolgus, rat and mouse is shown in Table16, where the aligned epitope sequence is underlined

TABLE 16  Comparison of IL12Rbeta1 antibodies epitope sequence in human,cynomolgus, rat and mouse Species Amino acid sequence HumanGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVD CynoGAMGQEKCYHITIFASAHPKKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVD Murine-TLEQEECYRITVFASKNPKNPMLWATVLSSYYFGGNASRAGTPRHVSVRNQTGDSVSVE Rat-TLDQEECYRITVFASKNPKNPMMWATVLSSYYFGGNVSRVGTPRHVSVRNHTEDSVSVE

The cynomolgus and human sequences only differ by one amino acid residuein this epitope sequence, which accounts for the cross reactivity of theantibodies with both human and cynomolgus sequences. In contrast, therodent sequences in this epitope show greater differences compared tothe human and cynomolgus sequences, this being consistent with the lackof specificity of the antibodies with the rodent IL12Rbeta1 proteins.

The invention claimed is:
 1. An isolated antibody or a protein with anantigen-binding portion of an antibody, comprising (a) a variable heavychain amino acid sequence comprising HCDR1 of SEQ ID NO:7, HCDR2 of SEQID NO:8, HCDR3 of SEQ ID NO:9 and a variable light chain amino acidsequence comprising LCDR1 of SEQ ID NO:10, LCDR2 of SEQ ID NO:11 andLCDR3 of SEQ ID NO:12; (b) a variable heavy chain amino acid sequencecomprising HCDR1 of SEQ ID NO:13, HCDR2 of SEQ ID NO:14, HCDR3 of SEQ IDNO:15 and a variable light chain amino acid sequence comprising LCDR1 ofSEQ ID NO:16, LCDR2 of SEQ ID NO:17 and LCDR3 of SEQ ID NO:18; or (c) avariable heavy chain amino acid sequence comprising HCDR1 of SEQ IDNO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21 and a variable lightchain amino acid sequence comprising LCDR1 of SEQ ID NO:22, LCDR2 of SEQID NO:23 and LCDR3 of SEQ ID NO:24; wherein said antibody or proteinbinds to the IL12Rβ1 polypeptide of SEQ ID NO:89.
 2. The isolatedantibody or protein of claim 1, wherein said antibody or proteincross-reacts with cynomolgous IL12Rβ1 polypeptide of SEQ ID NO:98. 3.The isolated antibody or protein of claim 1 wherein said antibody orprotein inhibits IL12 dependent IFN-γ production in human blood cellswith an IC₅₀ of 1 nM or less.
 4. The isolated antibody or proteinaccording to claim 1, which is fully human.
 5. The isolated antibody orprotein according to claim 1, which comprises a mutant or chemicallymodified amino acid Fc region, wherein said mutated or chemicallymodified Fc region confers no or decreased ADCC activity to saidantibody when compared to a corresponding antibody with wild type Fcregion.
 6. The isolated antibody or protein according to claim 1,comprising a V_(H) polypeptide sequence having at least 95 percentsequence identity to SEQ ID NO:51 and a V_(L) polypeptide sequencehaving at least 95 percent sequence identity to at least one of SEQ IDNO:52; (b) a V_(H) polypeptide sequence having at least 95 percentsequence identity to SEQ ID NO:53 and a V_(L) polypeptide sequencehaving at least 95 percent sequence identity to at least one of SEQ IDNO:54; or (c) a V_(H) polypeptide sequence having at least 95 percentsequence identity to SEQ ID NO:55 and a V_(L) polypeptide sequencehaving at least 95 percent sequence identity to at least one of SEQ IDNO:56.
 7. The isolated antibody or protein of claim 6, comprising aV_(H) polypeptide sequence and a V_(L) polypeptide sequence selectedfrom: a) SEQ ID NO: 51 and SEQ ID NO:52; b) SEQ ID NO: 53 and SEQ IDNO:54; and c) SEQ ID NO:55 and SEQ ID NO:56.
 8. The isolated antibody orprotein according to claim 7, comprising (a) heavy chain amino acidsequence of SEQ ID NO:58 and light chain amino acid sequence of SEQ IDNO:70; (b) heavy chain amino acid sequence of SEQ ID NO:62 and lightchain amino acid sequence of SEQ ID NO:70; (c) heavy chain amino acidsequence of SEQ ID NO:66 and light chain amino acid sequence of SEQ IDNO:70; (d) heavy chain amino acid sequence of SEQ ID NO:59 and lightchain amino acid sequence of SEQ ID NO:71; (e) heavy chain amino acidsequence of SEQ ID NO:63 and light chain amino acid sequence of SEQ IDNO:71; (f) heavy chain amino acid sequence of SEQ ID NO:67 and lightchain amino acid sequence of SEQ ID NO:71; (g) heavy chain amino acidsequence of SEQ ID NO:60 and light chain amino acid sequence of SEQ IDNO:72; (h) heavy chain amino acid sequence of SEQ ID NO:64 and lightchain amino acid sequence of SEQ ID NO:72; (i) heavy chain amino acidsequence of SEQ ID NO:68 and light chain amino acid sequence of SEQ IDNO:72; (j) heavy chain amino acid sequence of SEQ ID NO:91 and lightchain amino acid sequence of SEQ ID NO:70; (k) heavy chain amino acidsequence of SEQ ID NO:92 and light chain amino acid sequence of SEQ IDNO:71; or (l) heavy chain amino acid sequence of SEQ ID NO:93 and lightchain amino acid sequence of SEQ ID NO:72.
 9. A composition comprisingan antibody or protein according to claim 8, in combination with one ormore of a pharmaceutically acceptable excipient, diluent or carrier. 10.A composition comprising an antibody or protein according to claim 7, incombination with one or more of a pharmaceutically acceptable excipient,diluent or carrier.
 11. A composition comprising an antibody or proteinaccording to claim 1, in combination with one or more of apharmaceutically acceptable excipient, diluent or carrier.